Datasets:

---

rntc

/

tt-pp-cmco

like 0

Chemical protein synthesis is a well-recognized strategy to produce proteins that would otherwise be difficult or impossible to obtain, such as posttranslationally modified and mirror-image proteins, for biochemical and biomedical research ( 1 – 7 ). The strategy commonly requires the assembly of peptide segments to form a full-length protein via techniques such as native chemical ligation (NCL) ( 8 – 10 ), ketoacid-hydroxylamine ligation (KAHA ligation) ( 11 , 12 ), and serine/threonine ligation (STL) ( 13 , 14 ), which have greatly advanced the field. In peptide ligation reactions, the choice of solvent is critical, as the solvent must effectively dissolve the peptide segments at the desired concentration (e.g., ~1 mM for NCL) and simultaneously expose their reactive moieties for ligation ( 5 ). NCL is typically conducted in neutral aqueous solutions containing chaotropic agents such as guanidinium chloride (Gn·HCl) or urea ( 1 , 8 ), with organic solvents like dimethyl sulfoxide (DMSO)/H 2 O ( 12 ) and pyridine (Py)/acetic acid ( 13 , 14 ) often used for KAHA ligation and STL, respectively. Besides, both KAHA and STL work under acidic conditions. These solvents can usually work well for the proteins composed of well-behaved hydrophilic peptides ( 15 – 19 ), but recent studies have highlighted the increasingly often encountered challenges associated with the ligation of “difficult” peptides, which are either too poorly soluble to reach the millimolar concentrations required or prone to form aggregates or other undesired secondary structures that may mask the ligation sites ( 20 – 28 ).

39018393_p0

39018393

INTRODUCTION

biomedical

Study

en

Trifluoroacetic acid (TFA), one of the most powerful and most commonly used solvents in peptide chemistry ( 29 – 32 ), can effectively dissolve virtually all peptides and prevent the formation of unwanted secondary structures or aggregates ( 5 , 6 , 23 , 33 – 35 ). We envisioned that TFA, if it can be used as the ligation solvent, could address the long-standing and fundamental challenge presented by the ligation of difficult proteins, thereby enabling more general and robust chemical synthesis of all families of proteins. In this context, we report a TFA-assisted peptide conjugation followed by intramolecular native chemical ligation (TAL) method, which entails the chemoselective condensation of any family of peptide segments .

39018393_p1

39018393

INTRODUCTION

biomedical

Study

en

In our recent study on the development of substrates for STL, we found that 1,3-propanedithiol can react rapidly with a peptide salicylaldehyde ester in TFA to form an S,S -propanedithioacetal ( 36 ). Inspired by this finding, we designed a peptide thiosalicylaldehyde thioester ( 1a ) and a 1,3-dithiol-containing Cys-peptide ( 2a ) and attempted their ligation in TFA. Peptide 1a was prepared from the corresponding peptide hydrazide by oxidation with NaNO 2 ( 37 ), followed by thiolysis and acetal hydrolysis . Peptide 2a bearing a removable 1,3-dithiol auxiliary group was also readily synthesized using our previously developed removable backbone modification (RBM) strategy ( 38 ).

39018393_p2

39018393

The enhanc ed native chemical ligation by peptide conjugation in TFA

biomedical

Study

en

We dissolved 1a (10 mM) and 2a (10 mM) in TFA at room temperature. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis showed that 1a and 2a were completely and clearly consumed within only 30 s, accompanied by the formation of a new single peak 3a (97% HPLC yield), the corresponding molecular mass (MM) of which was consistent with that of the thioacetal intermediate . The TFA solution was then concentrated by N 2 gas blowdown and precipitated with cold diethyl ether to give solid 3a , which was used without purification. 3a (0.1 mM) was dissolved in a neutral phosphate buffer (H 2 O/CH 3 CN, v/v = 1:1; 20 mM phosphate, pH 7) to quantitatively afford 4a (97% HPLC yield) within 1 min. The observed MM of 4a was consistent with the theoretical MM of the ligated peptide , and it was stable in the presence of 5% hydrazine for 30 min , suggesting that the thioester moiety in 3a had been converted to the amide bond of 4a . Last, 4a was dissolved in a TFA cocktail (TFA/PhOH/dithiothreitol (DTT)/H 2 O/thioanisole, 87.5/2.5/2.5/5/2.5, v/w/w/v/v) to remove the auxiliary (Aux) group and generate 5a within 2 hours (HPLC yield of 94%). The observed MM of 5a was consistent with that of the target product . The isolated yield of the final product 5a was 66% from 1a , further evidence of the feasibility and good efficiency of the TAL method.

39018393_p3

39018393

The enhanc ed native chemical ligation by peptide conjugation in TFA

biomedical

Study

en

In a control experiment, peptides 1a (1 mM), 2a (1 mM), and 2e (1 mM) were dissolved together in TFA. As expected, 1a only reacted with 2a , but not with 2e , demonstrating that the aldehyde group of 1a chemoselectively reacts with the 1,3-dithiol of 2a in TFA, while the N-terminal Cys and the thiol group on the adjacent Cys remained inert. Comparison of the HPLC traces of 3a and the ligation product of thioester 1q with 2a showed no detectable racemization at the ligation site . The effect of the concentration of 1a and 2a in TFA on reaction time was also investigated : A reaction time of 30 s was sufficient to obtain HPLC yields of 97% and 98% at peptide concentrations of 100 mM and 10 mM, respectively, whereas 3 min and 30 min were required for HPLC yields of 97% at both 1 mM and 0.1 mM, respectively.

39018393_p4

39018393

The enhanc ed native chemical ligation by peptide conjugation in TFA

biomedical

Study

en

Conversion of 3a to 4a can be carried out with a wide range of peptide concentrations from 0.001 to 1 mM and a broad pH range from 5 to 9, all with high HPLC yields . In addition, quantitative conversion of 3a to 4a (HPLC yields more than 95%) can be readily performed in various solvent conditions. This includes denaturing aqueous solutions containing agents such as guanidine hydrochloride (Gn·HCl) or urea, as well as various combinations of water (H 2 O) with organic cosolvents such as acetonitrile (CH 3 CN), Py/acetic acid mixture, dimethylformamide (DMF), DMSO, hexafluoroisopropanol, and 2,2,2-trifluoroethano.

39018393_p5

39018393

The enhanc ed native chemical ligation by peptide conjugation in TFA

biomedical

Study

en

To investigate how the performance of TAL was affected by the location of the 1,3-dithiol modifications, the 1,3-dithiol group was introduced onto the amide between Lys 4 -Phe 5 or Gly 8 -Ala 9 sites in 2 to generate peptides 2b and 2c , respectively. 2b and 2c were separately ligated with 1a in TFA to afford 3b and 3c , which were converted to the corresponding 4b and 4c by treatment with phosphate buffer (0.1 mM, H 2 O/CH 3 CN, v/v = 1/1; 20 mM phosphate, pH 7) for 1 min and then advanced to the desired product 5a in HPLC yields of 94% and 93%, respectively, by exposure to TFA cocktails (TFA/PhOH/DTT/H 2 O/thioanisole, 87.5/2.5/2.5/5/2.5, v/w/w/v/v) for 2 hours . These results show that the TAL method can tolerate different sites for the installation of the 1,3-dithiol group.

39018393_p6

39018393

The enhanc ed native chemical ligation by peptide conjugation in TFA

biomedical

Study

en

The scope of the TAL method was assessed via the ligation in TFA between 2a and a series of model peptide thiosalicylaldehyde thioesters ( 1b to 1p ) bearing various C-terminal amino acids and having the general sequence GALKFERG X . The desired ligated peptides 5b to 5p except 5k were obtained with >90% HPLC yields ( Table 1 , entries 2 to 16); 5k , a peptide formed by the ligation of the highly sterically hindered Pro and Cys residues, formed in 85% HPLC yield ( Table 1 , entry 11). Moreover, 1a and 1p could ligate in TFA with 2d , bearing an N-terminal Gly(Aux), to generate the desired target products 5r and 5s . These results show that the TAL method is highly tolerant of sterically hindered amino acids at the C-terminal and N-terminal sites.

39018393_p7

39018393

The enhanc ed native chemical ligation by peptide conjugation in TFA

biomedical

Study

en

In previous work, we found that the peptide thioester Hin-Lig (1-27)-MPAA 6a derived from Haemophilus influenzae DNA Ligase (Hin-Lig) failed to ligate with Hin-Lig (28-85) 7a in phosphate buffer (pH 6.5) containing 6 M Gn·HCl, a denaturing solvent often used for NCL, because of the formation of colloidal particles ( 27 ). Although 6a and 7a did dissolve in the Gn·HCl-containing buffer, only hydrolytic by-product 6b , but not ligation product 8 was detected . An aqueous 6 M Gn·HCl solution of 6a or a mixture of 6a and 7a illuminated with a 530-nm laser pointer exhibited an obvious Tyndall effect, indicating the formation of colloidal particles of 6a , an effect also observed when 6a was dissolved in DMF/oxalic acid and Py/acetic acid, solutions that are commonly used for KAHA ligation and STL, respectively . In comparison, no Tyndall effect was detected for TFA solutions of 6a and 7a , indicating its ability to prevent the formation of undesired structures of 6a .

39018393_p8

39018393

The TAL method enabled the assembly of peptides prone to form colloidal particles

biomedical

Study

en

Peptide thioester Hin-Lig (1-27)–TSAL thioester 6 and Hin-Lig [28-85, L 35,Aux(LA) ] 7 were prepared from the corresponding peptide hydrazide and via a standard Fmoc-SPPS–based RBM strategy , respectively. Both peptides 6 (5 mM) and 7 (5 mM) easily dissolved in TFA to give a solution that did not exhibit a Tyndall effect. When peptides 6 (18 mg, 5 mM) and 7 (36 mg, 5 mM) were dissolved together in TFA, HPLC analysis showed that the reaction was complete within 10 min to give 8a , electrospray ionization mass spectrometry (ESI-MS) analysis of which showed it to have a MM consistent with that of the peptide dithioacetal thioester . Posttreatment (comprising precipitation with chilled ethyl ether, treatment with weekly acidic buffer for 1 hour, and cleavage with TFA cocktails for 2 hours) of 8a resulted in the production of the final product 8 (32% isolated yield starting from peptide 6 ).

39018393_p9

39018393

The TAL method enabled the assembly of peptides prone to form colloidal particles

biomedical

Study

en

In summary, TFA shows better solubilizing and denaturing properties than solvents traditionally used for peptide ligation and allows the efficient ligation of peptide segments refractory to canonical native chemical ligation.

39018393_p10

39018393

The TAL method enabled the assembly of peptides prone to form colloidal particles

biomedical

Study

en

To evaluate the potential of TAL to synthesize challenging proteins, we chose the single, 75-amino acid transmembrane protein severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Envelope (E) protein as a target, which is implicated in viral budding, release, and the host inflammatory response ( 39 ). A synthesis of E protein would be useful because of its importance as a target for antiviral drugs and to support the study of how posttranslational modifications such as acetylation and glycosylation regulate its functions.

39018393_p11

39018393

The TAL method promoted the chemical synthesis of membrane protein

biomedical

Study

en

Initially, we applied the conventional NCL strategy to prepare the E protein by dividing it into two segments between Leu 39 and Cys 40 . Because of the hydrophobic nature of these segments, we temporarily installed a solubilizing Lys 8 -tag on the amide bond of Phe 4 and Ile 46 to give E (1-39, F 4,Aux )–MPAA 9a and E (40-75, I 46,Aux ) 10a . Peptides 9a and 10a were successfully prepared via an Fmoc-SPPS–based RBM strategy in isolated yields of 44% and 33%, respectively. NCL of 9a and 10a was attempted by dissolving them in phosphate buffer (pH 7.0) containing 6 M Gn·HCl at 1 mM. ESI-MS analysis showed that no ligation product was formed; instead, 9a was completely hydrolyzed to form carboxylic acids 9b and 9c . Further examination showed that a Gn·HCl solution of 9a exhibited an obvious Tyndall effect, suggesting it to have formed colloidal particles . Therefore, although the RBM strategy can increase the solubility of hydrophobic peptides, it may not be sufficient to disrupt the formation of colloid structures, even in the presence of 6 M Gn·HCl.

39018393_p12

39018393

The TAL method promoted the chemical synthesis of membrane protein

biomedical

Study

en

We turned to the TAL method to address this challenge. Two peptides, E (1-39, F 4,Aux )–TSAL thioester 9 and E [40-75, I 46,Aux(LA) ] 10 were synthesized with isolated yields of 18% and 16% . To our delight, both 9 (2 mM) and 10 (2 mM) were completely dissolved in TFA at a concentration of 2 mM without any detectable Tyndall effect and underwent ligation within 10 min by RP-HPLC analysis to generate 11a , which was isolated as a solid pellet after concentration of the reaction mixture by N 2 blowdown and precipitation with cold ethyl ether. Treatment of 11a with a weakly acidic (pH 5.6) buffer for 1 hour and hydrazine hydrate (NH 2 NH 2 /solvent = 1/100, v/v, pH 8 to 9) for 0.5 hours gave 11b , which was dissolved in an HCl cocktail [1 M HCl/hexafluoroisopropanol and 5% triisopropylsilane (TIPS)] for 2 hours to generate the final product 11 with an overall yield of 9% starting from 9 . 11 was too hydrophobic for HPLC and was therefore analyzed by tricine–SDS–polyacrylamide gel electrophoresis (SDS-PAGE) instead; a clear band shift for 11 compared to its precursor 11b was observed. ESI-MS analysis of 11 showed that the observed MM was consistent with the theoretical MM . Last, the folding of 11 was performed in dodecylphosphocholine (DPC) solution (100 mM DPC, 20 mM sodium phosphate, 50 mM NaCl, pH 5.5, 30°C). The circular dichroism (CD) spectrum showed two representative negative peaks at 208 nm and 222 nm corresponding to structural features of α helices, consistent with the previously reported CD spectrum of recombinant E protein ( 39 ).

39018393_p13

39018393

The TAL method promoted the chemical synthesis of membrane protein

biomedical

Study

en

Next, we used TAL to synthesize nanobodies (Nbs) composed of nine β strands. Nbs, which consist of the smallest naturally available antigen-binding VHH domains of antibodies but lack light chains, are a class of antibodies demonstrating improved tissue penetration and water solubility compared with traditional antibodies while retaining traditional antibody-like antigen-binding affinity and stability ( 40 – 42 ). However, recent studies have identified challenges in chemical synthesis of Nbs due to the inefficient ligation of β strand–rich peptides ( 43 , 44 ).

39018393_p14

39018393

The TAL method allowed multiple-segment ligation to give nanobodies

biomedical

Study

en

We chose a 120-residue Nb that targets green fluorescent protein as a representative example ( 45 ). First, we tried the canonical ligation strategy. Nb was divided into three segments: Nb (1-52)–MPAA 12a , Nb (53-94)-NHNH 2 13a , and Nb (95-120) 14a , which were synthesized with isolation yields of 29%, 21%, and 41%, respectively . We attempted to assemble these segments in an N-to-C direction. Peptides 12a and 13a were dissolved in the phosphate buffer (pH 7.0) containing 6 M Gn·HCl, without observable turbidity. However, HPLC and ESI-MS analyses revealed that only a small fraction (~5%) of the ligation product 15c was produced, with most of the 12a converted to its corresponding hydrolyzed by-product 12b and intramolecular cyclization by-product 12c . We then discovered that 6 M Gn·HCl solutions of either 12a , 13a , or a mixture of the two all exhibited obvious Tyndall effects , indicating that both peptides had formed colloid structures.

39018393_p15

39018393

The TAL method allowed multiple-segment ligation to give nanobodies

biomedical

Study

en

We turned to use the TAL method to achieve the chemical synthesis of Nb. Three peptides Nb (1-52, C 21,Acm )–TSAL thioester 12 , Nb [53-94, F 67,Aux(LA) ]-NHNH 2 13 , and Nb [95-120, Q 109,Aux(LA) ] 14 were prepared. Peptide 12 was prepared with an isolated yield of 18% from the corresponding peptide hydrazide Nb (1-52, C 21,Acm )–NHNH 2 , in which the Cys 21 was temporarily protected by an Acm group . Peptides 13 and 14 bearing the 1,3-dithiol group at Phe 67 and Glu 109 were synthesized with isolated yields of 11% and 18%, respectively . Peptides 12 (2 mM), 13 (2 mM) and 14 (2 mM) were all fully soluble in TFA without evidence of any Tyndall effect, suggesting that TFA effectively inhibits the formation of peptide colloidal particles .

39018393_p16

39018393

The TAL method allowed multiple-segment ligation to give nanobodies

biomedical

Study

en

The assembly of full-length Nb was carried out in an N-to-C direction by two successive peptide ligations in TFA. First, 12 (2 mM) and 13 (2.1 mM) were dissolved in TFA, and the peptide dithioacetal thioester 15a was formed within 5 min at 30°C, as confirmed by ESI-MS (observed: 10,459.90 Da; calculated: 10,460.30 Da). Solid 15a was isolated after concentration and precipitation and then treated with weakly acidic H 2 O/CH 3 CN (v/v = 1/1) buffer (0.1 mM, Py/acetic acid, pH 5.6) to the backbone amide and TFA cocktails (TFA/TIPS/H 2 O, 95/2.5/2.5, v/v/v) to remove the auxiliary group ( 46 ). The resulting peptide underwent free-radical desulfurization by treatment with TECP/VA-044 to give the product 15d , which was purified by HPLC (42% isolated yield starting from peptide 12 ), and its molecular mass verified by ESI-MS . Last, 15d was converted to peptide thioester 15 in 44% isolated yield by the oxidation/thiolysis protocol.

39018393_p17

39018393

The TAL method allowed multiple-segment ligation to give nanobodies

biomedical

Study

en

Peptides 15 (1 mM) and 14 (1.2 mM) were ligated by mixing their respective TFA solutions. The ligation was complete within 5 min at 30°C to afford 16a , the molecular mass of which was consistent with that of the expected peptide dithioacetal thioester (observed MM: 14,797.21 Da; theoretical MM: 14,797.53 Da). After concentration and precipitation, 16a was treated with a weakly acidic H 2 O/CH 3 CN (v/v = 1/1) buffer (0.1 mM, Py/acetic acid, pH 5.6) and the TFA cocktails (TFA/PhOH/DTT/H 2 O/thioanisole, 87.5/2.5/2.5 /5/2.5, v/w/w/v/v), followed by PdCl 2 (50 equiv.) to remove the Acm protecting group. Full-length Nb protein 16d was purified by HPLC with an isolated yield of 44% starting from 15 and was verified by ESI-MS .

39018393_p18

39018393

The TAL method allowed multiple-segment ligation to give nanobodies

biomedical

Study

en

Purified 16d (0.2 mg/ml) was dissolved in an aqueous buffer [6 M Gn·HCl and 100 mM tris (pH 8.5)] at 4°C for 48 hours to form the intramolecular disulfide bond. Gn·HCl was removed by gradient dialysis. Subsequent purification by size exclusion chromatography yielded the final folded Nb 16 (isolation yield: 62%), which was characterized by ESI-MS and tricine–SDS-PAGE . CD analysis revealed that synthetic Nb 16 exhibited structural features including β sheets characteristic of recombinant Nb . We evaluated the binding of green fluorescent protein (GFP) and Nb by titrating purified wild-type GFP (WT GFP) with various concentrations (0, 5, 10, 25, or 50 nM) of either synthetic Nb 16 or recombinant Nb. Both synthetic and recombinant Nb induced fluorescence enhancement, and ca. fourfold enhancement of fluorescence intensity was observed when the concentration of Nb reached 50 nM, consistent with the previous report ( 43 , 45 ).

39018393_p19

39018393

The TAL method allowed multiple-segment ligation to give nanobodies

biomedical

Study

en

In summary, we have discovered that TFA can serve as an effective solvent for ligating virtually any peptide segments, including those that are poorly soluble in solvents like 6 M Gn·HCl aqueous buffer and/or prone to form colloid structures. In combination with native chemical ligation, this peptide conjugation strategy in TFA can conceptually address the long-standing and fundamental challenge presented by the ligation of increasingly often encountered difficult proteins, thereby enabling more general and robust chemical synthesis of proteins. The TAL method is based on the selective and rapid reaction between a peptide thiosalicylaldehyde thioester and a 1,3-dithiol–containing peptide in TFA to give a thioacetal intermediate that can be easily converted to the final ligation product by a simple posttreatment involving concentration, precipitation, and treatment with buffer and TFA cocktails, with a similar as the prior thiol capture method and templated ligation ( 47 , 48 ). The scope of the TAL method was studied via the condensation of numerous model peptides, and its utility and practicality were exemplified by the successful preparation of several challenging proteins, including the E protein and an Nb. Collectively, these results establish the TAL method as a general, robust, and potentially invaluable method for the ligation of peptides and would expand the range of proteins now accessible by chemical synthesis. Studies to develop new types of TFA-compatible ligation reactions and optimize the auxiliary groups used to install TFA-compatible reactive moieties onto peptides are ongoing in our laboratories and are expected to lead to a conceptually new category of peptide ligation technology; the results will be reported in due course.

39018393_p20

39018393

DISCUSSION

biomedical

Study

en

Rink amide resin and 2-Cl-Trt-Cl resin were bought from Nankai Hecheng Science & Technology Co., Ltd (Tianjin, China). Trityl-OH ChemMatrix resin and rink amide ChemMatrix resin were bought from PCAS BioMatrix Inc. The peptide synthesis tubes were bought from Synthware Glass Co. Ltd. All Fmoc amino acids and Boc amino acids were bought from GL Biochem (Shanghai, China). O -(7-azabenzotriazol-1-yl)- N , N , N ′, N ′-tetramethyluronium hexafluorophosphate (HATU) and O -(6-chlorobenzotriazol-1-yl)- N , N , N ′, N ′-tetramethyluronium 10 hexafluorophosphate (HCTU) were bought from GL Biochem (Shanghai, China). N , N ′-diisopropyl-carbodiimide (DIC), ethyl cyanoglyoxylate-2-oxime (Oxyma), N , N -diisopropylethylamine (DIPEA), di- tert -butyl decarbonate, 4-dimethylaminopyridine (DMAP), N -Boc–γ-aminobutyric acid (Boc-GABA-OH), lipoic acid (LA), sodiumborohydride (NaBH 4 ), tetrahydroxydiboron [B 2 (OH) 4 ], 4,4′-bipyridine,4-mercaptophenylacetic acid (MPAA), tris(2-chloroethyl) phosphate (TCEP), trifluoroacetic acid (TFA), and triisopropylsilane were bought from Energy-Chemical (Shanghai, China). Pyridine, acetic acid (AcOH), 1,2-ethanedithiol (EDT), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), trifluoroethanol (TFE), and thioanisole were bought from J&K Scientific (Beijing, China). Guanidine hydrochloride (Gn·HCl), sodium phosphate monobasic (Na 2 HPO 4 ), N , N -dimethylformamide (DMF), dichloromethane (DCM), acetonitrile (CH 3 CN), methanol, anhydrous diethyl ether, hydrochloric acid (HCl), hydrazine hydrate aqueous solution (NH 2 NH 2 ·H 2 O), di- tert -butyl decarbonate (Boc 2 O), acetic anhydride (Ac 2 O), and phenol were bought from Sinopharm Chemical Reagent.

39018393_p21

39018393

Reagents and materials

biomedical

Other

en

Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on a Shimadzu Prominence HPLC System (Shimadzu Corp., Japan). Peptide analysis was performed on a YMC C4 (4.6 mm × 250 mm) column at a flow rate of 1.0 ml/min, and a YMC C4 (10 mm × 250 mm or 22 mm × 150 mm) column at a flow rate of 4.0 or 6.0 ml/min was used for peptide purification. Buffer A (0.1% TFA in water), buffer B (0.08% TFA in CH 3 CN solution), and buffer C [0.08% TFA in the mixed solvents iPrOH-CH 3 CN (v/v, 1:1)] were used as the solvents. The solvent gradient was optimized for each peptide.

39018393_p22

39018393

High-performance liquid chromatography, mass spectrometry, and circular dichroism spectroscopy

biomedical

Study

en

A Shimadzu Prominence HPLC System (Shimadzu Corp., Japan) with LCMS-2020 was used to record electrospray ionization mass spectrometry (ESI-MS) spectra. Applied Photophysics Pistar Π-180 circular dichroism spectrometer was used to record circular dichroism spectra.

39018393_p23

39018393

High-performance liquid chromatography, mass spectrometry, and circular dichroism spectroscopy

biomedical

Study

en

All peptides were synthesized by using an automated microwave peptide synthesizer (CEM Liberty Blue). Rink amide AM resin was used to prepare C-terminal amide peptides, while 2-Cl-Trt-NHNH 2 resin was used to yield Cterminal hydrazide peptides. The resin was swelled in a mixture of DCM and DMF (5 ml + 5 ml) for 30 min. All other amino acids were coupled using the standard microwave-assisted double coupling process. Each cycle involved a Fmoc deprotection step using 20% piperidine in DMF (1 min at 90°C) and an amino acid coupling step using a fourfold excess of 0.2 M Fmoc-protected amino acid, 0.5 M DIC, and 1.0 M Oxyma in DMF (twice for 10 min at 50°C for His and 2 min at 90°C for other residues). After peptide assembly, TFA cocktails (TFA/phenol/thioanisole/EDT/H 2 O, 85/5/5/2.5/2.5, v/w/v/v/v) were used to cleavage peptides from the resin. The cleavage solution was collected and then concentrated by N 2 blowing. The crude peptide was subsequently precipitated with cold diethyl ether and separated by centrifugation. Further analysis and purification were performed using RP-HPLC and ESI-MS.

39018393_p24

39018393

Automated microwave peptide synthesis

biomedical

Study

en

The peptide assembly was performed using an automated microwave peptide synthesizer. After the removal of the Fmoc group of the amino acid that needs to be connected to the Aux, the remaining process requires manual handling, 2-hydroxy-4-methoxy-5-nitrobenzaldehyde [4 equivalent (equiv.)] in DMF was added to the resin and incubated for 60 min. Subsequently, NaBH 4 (5 equiv.) in DMF was added to the resin for 5 min (twice). Thereafter, the resin was washed with the following solvents: DMF, H 2 O, methanol, DCM, and DMF, respectively (five times).

39018393_p25

39018393

Synthesis of auxiliary-containing peptides

biomedical

Study

en

The standard microwave-assisted double coupling protocol was used to assemble the following amino acids. Note that the amino group of the last amino acid should be protected with a Boc group for the subsequent Fmoc SPPS for Lys-tag or LA-tag.

39018393_p26

39018393

Synthesis of auxiliary-containing peptides

biomedical

Other

en

After the peptide chain assembly, B 2 (OH) 4 (20 equiv.) in DMF (4 ml for 0.1 mmol resin) and 4,4′-bipyridine (0.25 equiv.) in DMF (1 ml for 0.1 mmol of resin) were added to the resin and reacted for 20 min (twice). This step transformed the nitro into the free amino group.

39018393_p27

39018393

Synthesis of auxiliary-containing peptides

biomedical

Study

en

For the introduction of a LA-tag, LA coupling was carried out by adding a solution of the LA (10.0 equiv.), HATU (9.8 equiv.), and DIEA (20 equiv.) in DMF to the resin for 1 hour (twice) at 30°C. In the case of Lys 8 -tag, a solution of Fmoc-Gly-OH (8.0 equiv.), HATU (7.6 equiv.), DMAP (0.8 equiv.), and DIEA (16.0 equiv.) in DMF were added to the resin for 1 hour (two times) at 30°C for the coupling of Fmoc-Gly-OH, followed by standard double coupling using the microwave method to introduce seven Fmoc-Lys(Boc)-OH and one Boc-Lys(Boc)-OH, respectively. For the LA-Lys 6/8 –tag, a solution of Fmoc-Gly-OH (8.0 equiv.), HATU (7.6 equiv.), DMAP (0.8 equiv.), and DIEA (16.0 equiv.) in DMF were added to the resin for 1 hour (two times) at 30°C for the coupling of Fmoc-Gly-OH, followed by a standard double coupling using the microwave method to incorporate either six or eight Fmoc-Lys(Boc)-OH residues. Then, LA was carried out by adding a solution of the LA (10.0 equiv.), HATU (9.8 equiv.), and DIEA (20.0 equiv.) in DMF to the resin for 1 hour (twice) at 30°C. For all tags, the resin was treated with 10 ml of 20% (v/v) of piperidine solution (DMF) reacting for 60 min at room temperature to remove all the amino acids attached to the Aux and subsequently release the 2-OH group. Notably, because of the potential of piperidine to cleave the disulfide bond in LA, we need to add DIEA (0.5 ml) in DMF to the resin for 30 min to promote the reformation of the disulfide bond for LA-tag and LA-Lys 6/8 –tag.

39018393_p28

39018393

Synthesis of auxiliary-containing peptides

biomedical

Study

en

The 2-OH group of the auxiliary was then capped using Ac 2 O or Boc-GABA-OH as follows, which enabled the auxiliary groups to withstand TFA cleavage: (i) a solution of 10 ml of Ac 2 O/DIEA/DMF (1:1:8, v/v/v) to the resin for 30 min at 30°C; and (ii) Boc-GABA-OH (10 equiv.), DIC (10 equiv.), Oxyma (10 equiv.), and DMAP (1 equiv.) were dissolved in DMF and added to peptide resin for 20 min (two times) at 90°C. Last, a standard TFA cleavage step was carried out to remove the side chain protecting groups and release the auxiliary-modified peptides.

39018393_p29

39018393

Synthesis of auxiliary-containing peptides

biomedical

Study

en

Peptide hydrazide (1 equiv.) was dissolved in 0.2 M phosphate buffer containing 6 M Gn·HCl (pH 3.0) and was oxidized by adding NaNO 2 (10 equiv.) to the solution for 20 min at −15°C to generate the corresponding peptide azide. To convert peptide azide into its thioester, MPAA (30 equiv.) was then added to the solution for 10 min, and the pH was adjusted to 5~6. Upon completion of the thioester transfer, TCEP (50 equiv.; pH 5) was added to the solution for 10 min to reduce the reaction. Then, the pH of the solution was adjusted to 1, and the MPAA was extracted with chilled ether (three times) before the peptide was separated by RP-HPLC.

39018393_p30

39018393

Preparation of peptide-MPAA

biomedical

Study

en

Peptide hydrazide (1 equiv.) was dissolved in 0.2 M phosphate buffer containing 6 M Gn·HCl (pH 3.0) and was oxidized by adding NaNO 2 (10 equiv.) to the solution for 20 min at −15°C to generate peptide azide. To convert peptide azide into its thioester, 2-(1,3-dioxolan-2-yl) benzenethiol (30 equiv.) in CH 3 CN was then added to the solution for 10 min, and the pH was adjusted to 5~6. Notably, the volume ratio of aqueous buffer to CH 3 CN in the final reaction solution was 2 to 1. Upon completion of the thioester transfer, TCEP (50 equiv.; pH 5) was added to the solution for 10 min to reduce the reaction. Then, the pH of the solution was adjusted to 1, and the 2-(1,3-dioxolan-2-yl) benzenethiol was extracted with ice ether (three times) before the peptide was separated by RP-HPLC.

39018393_p31

39018393

Preparation of peptide-TSAL thioester

biomedical

Study

en

Peptide hydrazide (1 equiv.) was dissolved in 0.2 M phosphate buffer containing 6 M Gn·HCl (pH 3.0) and was oxidized by adding NaNO 2 (10 equiv.) to the solution for 20 min at −15°C to generate peptide azide. After that, the phosphate solution of MPAA (50 equiv.) and N-terminal Cys peptide (1 to 1.2 equiv.) was added for subsequent native chemical ligation (pH 6.5 to 7.0; 30°C). The ligation was tracked with analysis of RP-HPLC and ESI-MS. Once the ligation was accomplished, the ligation system was reduced by the addition of 0.1 M neutral TCEP 30 solution (100 equiv.) for 10 min.

39018393_p32

39018393

Native chemical ligation

biomedical

Study

en

The ligation of peptide-TSAL thioester (1 to 10 mM) and the 1,3-dithiol-containing peptide (1 to 10 mM) was carried out in TFA solution at room temperature. The ligation was tracked with analysis of RP-HPLC and ESI-MS. Upon completion of the ligation, the TFA solution was concentrated by blowing N 2 and precipitation with cold diethyl ether and then afforded peptide-TSAL-dithioacetal thioester. This intermediate is subsequently characterized by RP-HPLC and ESI-MS. Then, postligation treatment of peptide-TSAL-dithioacetal thioester (0.1 to 1 mM) was performed under phosphate buffer (H 2 O/CH 3 CN, v/v, 1/1; 20 mM phosphate; pH 5.5 to 7) solution and took place rapidly and quantitatively to give native amide peptide. Last, the solution can be directly freeze-dried for the preparation of the native backbone peptide by treatment with TFA cocktails to remove the Aux group within 1 to 4 hours at 30°C. The final product was purified by RP-HPLC, and its identity was verified by ESI-MS.

39018393_p33

39018393

The TAL method

biomedical

Study

en

The peptide was dissolved in ligation buffer (6 M Gn·HCl or TFA) in a glass vial. The solution was illuminated from the bottom of bottle using a laser pointer (~530 nm) to observe whether there was a bright laser beam. The observation of a bright laser beam led to the conclusion that the peptide had formed colloidal particles.

39018393_p34

39018393

The Tyndall effect

biomedical

Study

en

The peptide (1.0 μmol) was dissolved in the desulfurization buffer [700 μl; 6.0 M Gn·HCl, 0.2 M Na 2 HPO 4 , and 0.5 M TCEP (pH 6.9)]. Subsequently, 70 μl of tBuSH and 700 μl of VA-044 solution (0.1 M in water) were added, and the reaction mixture was stirred at 37°C for 3 to 24 hours. The reaction was monitored using RP-HPLC and ESI-MS.

39018393_p35

39018393

Free radical desulfurization

biomedical

Study

en

The peptide containing the Acm group (1 mM) was dissolved in the aqueous buffer [6.0 M Gn·HCl and 0.2 M Na 2 HPO 4 (pH 7.2)] and treated with PdCl 2 (50 equiv.) for 15 min at 30°C to remove the Acm group. Upon completion of the reaction, 200 equiv. of dithiothreitol (DTT) was added to quench the reaction and precipitate free palladium from the reaction mixture. After centrifugation, the supernatant solution was collected and purified using semipreparative HPLC.

39018393_p36

39018393

Removal of the Acm group

biomedical

Study

en

The auxiliary-containing peptide was dissolved in TFA cocktails (TFA/PhOH/DTT/H 2 O/thioanisole, 87.5/2.5/2.5/5/2.5, v/w/w/v/v) and incubated at 37°C for 1 to 5 hours to remove the auxiliary groups. Note that the auxiliary groups can also be cleaved using HFIP and 5% triisopropylsilane with either 0.1 or 1 M HCl. After the cleavage, the resulting solution was concentrated using N 2 blowing and precipitated with chilled diethyl ether to yield the target peptide that was separated by centrifugation and then purified by RP-HPLC. The insoluble crude solid powder that could not be purified by HPLC underwent an additional washing step with water three times to eliminate the watersoluble Lys-tag. The final product was analyzed by ESI-MS and/or tricine SDS–polyacrylamide gel electrophoresis.

39018393_p37

39018393

Removal of the auxiliary group

biomedical

Study

en

The chemically synthetic SARS-CoV-2 envelope (E) (0.2 mg/ml) was folded under 20 mM sodium phosphate (pH 5.5) (100 mM diphenylamine carboxylate and 50 mM NaCl) at 30°C for 24 hours.

39018393_p38

39018393

Folding of SARS-CoV-2 envelope

biomedical

Study

en

The full-length Nb (10 mg) was dissolved in 50 ml of aqueous buffer [6.0 M Gn·HCl and 100 mM tris (pH 8.5)] and incubated at 4°C for 2 days to form an intramolecular disulfide bond. The folding process was then carried out by stepwise dialysis to obtain folded Nb, which was subsequently purified by gel filtration chromatography using a Superdex 200 10/300 GL column (GE Healthcare) with tris buffer (100 mM; pH 8.5).

39018393_p39

39018393

Folding of Nb

biomedical

Study

en

The fluorescence binding assay was carried out by titrating 50 nM purified wild-type green fluorescent protein (WT GFP) with 0 to 50 nM of the chemically synthesized GFP Nb or the recombinant GFP Nb. The emission intensity of WT GFP was quantified using a laser scanner and a multifunction microplate reader (SpectraMax iD5, Molecular Devices; excitation, 470 nm; excitation, 510 nm).

39018393_p40

39018393

Fluorescence binding assay

biomedical

Study

en

The plasmid for expression was obtained by inserting the DNA sequences of E protein, WT GFP, or Nb into the pET-28a(+) plasmid using the restriction endonucleases Nco I and Xho I. GenScript Biotech (Nanjing, China) synthesized the genes for E protein, WT GFP, and Nb, which were then expressed and purified in the same manner. The BL21(DE3) Escherichia coli cells were transformed with the plasmid. The E. coli cells containing the expression vector were inoculated (1:100 dilution) into LB medium with kanamycin (100 μg/ml). Protein expression was induced with 1.0 mM isopropyl-β- d -thiogalactopyranoside at 37°C until the optical density at 600 nm reached 0.6 to 0.8. After isopropyl-β- d -thiogalactopyranoside treatment, the E. coli cells were able to express proteins at 18°C for 16 to 24 hours. The cells were then harvested and lysed by ultrasonication (30% power, 5-s on, 5-s off, 60 min) in 0.1 M tris buffer [0.5 M NaCl and 1 mM phenylmethylsulfonyl fluoride (pH 8.0)]. The supernatant obtained after centrifugation was purified using a Ni–nitrilotriacetic acid (NTA) column. The elution from the Ni-NTA purification was combined and further purified using size exclusion chromatography on a Superdex 200 10/300 column (GE Healthcare). Last, the purified WT GFP or Nb was concentrated, flash-frozen in liquid nitrogen, and stored at −80°C for future use. For E protein, the elution from the Ni-NTA purification was subjected to cleavage of the His-SUMO tag by treatment with Ulp1 protease and dialyzed in dialysis buffer [50 mM tris-HCl, 100 mM NaCl, and 2 mM DTT (pH 8.0)] at 4°C for 16 hours. The protein solution was filtered through Ni-NTA resin to collect the effluent, which was then purified by size exclusion chromatography to give the final product. Expressed sequences are as follows: E protein (His-SUMO-E protein), MGSSHHHHHHGSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG MYSFVSEEIGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV; WTGFP , MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKHHHHHH; Nb, MVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSHHHHHH.

39018393_p41

39018393

Cloning and purification of E protein, WT GFP, and Nb

biomedical

Study

en

Controlling droplets is crucial in various practical applications, spanning biomedical fields ( 1 , 2 ), chemical reactions ( 3 ), thermal regulation ( 4 ), water harvesting ( 5 ), and electronics ( 6 ). Various external stimuli, such as magnetic ( 7 , 8 ), electrical ( 9 , 10 ), optical ( 11 , 12 ), and ultrasound ( 13 , 14 ) are used to enhance the flexibility and precision of droplet manipulation. Thanks to substantial technological advancements in the past decade, we can now transport, merge, disperse, trap, and sort individual droplets at remarkably high throughput ( 15 – 17 ). The fluidity of droplets also offers vast design possibilities for miniature soft robots ( 18 , 19 ), promising breakthroughs in biomedical applications ( 20 – 22 ), particularly in accessing hard-to-reach sites of the human body. To address the limitations of conventional soft robots, particularly those based on magnetic elastomers ( 23 – 25 ), recent research has focused on droplet-based robots with enhanced deformation capabilities, such as liquid metal robots ( 26 – 30 ) and ferrofluid robots ( 31 – 34 ). For instance, a two-dimensional (2D) ferrobotic system using ferrofluid facilitates the measurement of active-matrix metallopeptidases (a cancer malignancy and inflammation biomarker) in human plasma and the detection of the SARS-CoV-2 virus in clinical samples ( 35 , 36 ). Another ferrofluid robotic system, using a 2D electromagnetic array, enables targeted drug delivery and delicate object manipulation ( 32 ). Magnetic slime robots, using non-Newtonian fluids, demonstrate versatility and on-the-fly reconfiguration capabilities ( 37 ). Liquid droplet–based robots with superior deformation capabilities can undergo extreme transformations, such as splitting and fusion, leading to increased flexibility and scalability ( 38 ).

39018413_p0

39018413

INTRODUCTION

biomedical

Review

en

However, current research primarily concentrates on the deformation and manipulation of individual droplets within a 2D plane ( 39 – 41 ). This focus not only constrains the potential application scenarios of droplets but also restricts the functionality and operational capabilities of droplet-based miniature soft robots ( 28 , 32 , 42 , 43 ). While 3D manipulation of droplets can be achieved using external tools such as magnetic cilia or magnetic tweezers ( 44 , 45 ), their passive motion dependent on such tools hinders adaptability to unstructured environments, thereby limiting their utility across various potential applications. Consequently, achieving 3D manipulation and active deformation of individual droplets stands as a crucial challenge in this field. Moreover, there has been limited exploration into the interactions among multiple droplets and their self-organizing behaviors ( 15 , 31 ), particularly concerning in situ manipulation and reconfiguration of multiple droplet collectives. This aspect holds practical significance as liquid droplets seldom exist in isolation but rather in clusters. Effectively regulating droplet collectives can enhance task efficiency of droplet-based robots and substantially augment the versatility of miniature robots.

39018413_p1

39018413

INTRODUCTION

biomedical

Study

en

In our study, we introduce a bimodal strategy for 3D manipulation of both individual droplets and droplet collectives. This strategy relies on two external fields: magnetic and light fields. Magnetic fields serve to modulate the morphology and orientation of individual droplets, facilitating their division into multiple sub-droplets and subsequent assembly into droplet collectives. By adjusting the parameters of the external magnetic field, diverse patterns of droplet collectives emerge, ranging from chain-like and layer-stacked structures to dispersed columnar formations. Furthermore, the application of light fields induces Marangoni flows within the droplets, and the droplets expand in volume, causing them to acquire upward lift. Through precise parameter adjustments of the light field, individual droplets can maneuver on a 2D plane, hover in 3D space, navigate directionally, and even traverse continuous obstacles. Notably, the light field not only influences the morphology of droplet collectives, enabling the assembly of dispersed columnar structures into higher pyramidal formations, but also grants them the capability of 3D movement and hovering. Illustrated in Fig. 1A , the control principle of droplets elucidates their operational versatility. Figure 1B showcases various functionalities of droplets, including traversing complex obstacles, self-assembling into collectives for levitation tasks, or acting as actuators in jellyfish-like structures, thereby achieving sunlight-driven locomotion. Compared to conventional ferrofluid droplets controlled solely by magnetic fields ( 31 – 36 , 46 – 48 ), the utilization of coupling fields in magnetic droplets yields more intricate motion patterns and collective behaviors. This advancement substantially broadens the potential applications of droplet-based robots within the realm of miniature soft robotics.

39018413_p2

39018413

INTRODUCTION

biomedical

Study

en

The coprecipitation method is used for synthesizing Fe 3 O 4 nanoparticles coated with oleic acid ( 49 – 51 ). Subsequently, these nanoparticles are incorporated into mineral oil, resulting in the formation of an oil-based ferrofluid . Fe 3 O 4 nanoparticles, coated with oleic acid, are uniformly dispersed in the mineral oil, exhibiting the capability to absorb near-infrared (NIR) light and convert it into heat energy via photothermal conversion. As depicted in fig. S2A, observations from an infrared camera reveal that upon directing an infrared laser at the surface of the ferrofluid from 10 cm, the surface temperature of the ferrofluid increases from room temperature (25°C) to 140°C in approximately 15 s. The magnitude of laser power influences the maximum temperature attained on the ferrofluid surface: At 3 W/cm 2 , the surface temperature reaches 70°C, whereas at 15 W/cm 2 , it escalates to 160°C . Snapshots captured by a high-speed camera depict that upon placing ferrofluid droplets in a water phase environment and subjecting them to laser irradiation (wavelength: 808 nm, intensity I : 24 W/cm 2 , direction D : 90° vertical), the droplets exhibit vertical upward movement from the bottom . Upon turning off the laser, the ferrofluid droplets promptly descend to the bottom. Volume expansion of the droplet under laser irradiation can be observed from the snapshots obtained by the high-speed camera. When the optical field is deactivated, the droplet deformation ceases to expand and returns to its original approximate spherical state. The relationship between the vertical position height and irradiation time changes of the ferrofluid droplet demonstrates the exceptional responsiveness of the droplet to laser irradiation. Specifically, upon laser activation, the ferrofluid droplets ascend from the base to a height of 12 mm within 0.5 s.

39018413_p3

39018413

Governing mechanism of 3D locomotion of individual ferrofluidic droplets

biomedical

Study

en

The working principle of NIR laser-induced droplet 3D locomotion involves two primary mechanisms. First, localized laser spot irradiation on the ferrofluid droplet leads to temperature elevation, resulting in uneven temperature distribution between the ferrofluid and water phases. This temperature differential triggers Marangoni flows, propelling the droplet upward. Second, the heat-induced expansion of tiny bubbles within the ferrofluid droplet causes an increase in volume, thereby enhancing buoyancy and facilitating upward movement (see more details in note S1). In both drive mechanisms, enhanced buoyancy dominates. We first analyze the uneven temperature-induced thermocapillary flow. Upon laser irradiation, the droplet’s surface experiences a gradual decrease in temperature from top to bottom due to gradual laser power absorption along the light path. Consequently, Marangoni convection arises inside and outside the droplet. Conservation of momentum dictates that the droplet moves in the opposite direction of the external Marangoni flow, resulting in upward floating. COMSOL simulation indicates that the maximum flow velocity of the Marangoni flow generated by the droplet is approximately 30 mm/s when a temperature difference of 30 K exists between the upper and lower surfaces of the droplet. In addition, the expansion of bubbles within the droplet also contributes to its floating behavior. COMSOL simulation and experimental verification support these two mechanisms . A permanent magnet fixes the ferrofluid droplet on the substrate, and tracer particles added to its surface allow observation of the trajectory of the particles under laser irradiation. This observation confirms the presence of Marangoni convection inside the droplet . Moreover, the expansion of the droplet’s volume is visibly observed during this process. Continued laser irradiation leads to droplet expansion and eventual splitting into two droplets. One droplet, attracted to the permanent magnet, remains fixed to the substrate, while the other, containing bubbles, continues to float upward and bursts upon reaching the water surface . Throughout the expansion process until splitting, the droplet’s maximum volume change rate is recorded as 2.58 times, indicating a corresponding increase in buoyancy compared to its initial state .

39018413_p4

39018413

Governing mechanism of 3D locomotion of individual ferrofluidic droplets

biomedical

Study

en

In addition to vertical upward floating motion, the light field enables hovering and translational motion of individual droplets in 3D space. We systematically study the influence of light intensity and droplet volume on the vertical movement of droplet. Our findings reveal that a light field intensity exceeding 36 W/cm 2 is required for upward motion of ferrofluid droplets of various volumes . In addition, we observe that light intensity dictates both the height and speed of droplet ascent, with stronger light fields resulting in higher and faster ascent rates . Furthermore, droplet size also plays a notable role, as larger droplets rise lower and slower under the same light field intensity . Notably, ambient temperature can affect the rate of droplet rise . Specifically, as the temperature increases from 5° to 45°C, the droplet rising speed gradually increases. However, beyond 45°C, the rise velocity decreases significantly. We attribute this phenomenon to the influence of ambient temperature on the heat dissipation process of the droplet, ultimately determining the temperature difference between the droplet and the environment, and thereby affecting its rise rate. As depicted in Fig. 3A and the corresponding schematic diagram in fig. S7A, irradiating the NIR laser horizontally from the side propels the droplet to continuously ascend. After 10 s, the droplet reaches the target position, and the height of the optical path is then set at 5.5 mm. Subsequently, the droplet consistently performs up-and-down floating motion near the set height, with the height difference between its upward and downward motion within the range of 0.5 mm (movie S3). Similarly, directing a NIR laser at an inclined angle to the ferrofluid droplet, approximately 30°, induces translational motion in 3D space, as demonstrated in Fig. 3B and the corresponding schematic diagram in fig. S7A. In this scenario, the ferrofluid droplet translates 10 mm at a height of 3.5 mm without contacting the substrate within 15 s (movie S3). 3D motion capability upon the droplets by the light field enables individual droplets to adapt to complex 3D environments effectively. For instance, a ferrofluid droplet monomer with 3D locomotion capability can navigate through maze environments filled with obstacles, akin to the game “Flappy Bird,” as illustrated in fig. S7B. As shown in fig. S7C, two ferrofluid droplets initially positioned at opposite ends of a vertical maze at t = 0 s traverse the vertical obstacles over 183 s under the influence of the light field, eventually converging at the maze’s center.

39018413_p5

39018413

Motion control of individual droplets

biomedical

Study

en

When both the magnetic field and the light field are simultaneously applied, the droplet’s mode can be adjusted during 3D motion. As depicted in Fig. 3C , under a vertical static magnetic field (with a magnetic field strength B of 9 mT and an angle α of 90° between the magnetic field and the horizontal plane), the ferrofluid droplet adopts a vertical shuttle shape. Subsequently, when a vertical light field is applied, the droplet maintains this shuttle shape while rising vertically (movie S4). Alternatively, under a static magnetic field, altering the angle α to 0° or 45° results in the ferrofluid droplet maintaining a horizontal shuttle or an inclined shuttle shape, respectively, before ascending vertically . Furthermore, in addition to the static magnetic field, a dynamic magnetic field can also be used. As illustrated in Fig. 3D , driven by the dynamic magnetic field coupled with the optical field, the ferrofluid droplet ascends while executing a kayaking maneuver (movie S4). Programmable adjustments to the dynamic magnetic field allow for vertical uplift while performing rotational, tumbling, and stretching motions . By periodically switching the light field on and off, ferrofluid droplets can rise and fall cyclically . However, the maximum height reached by the ferrofluid droplet under the dynamic magnetic field is greater. This is attributed to the droplet continually changing shape under the dynamic magnetic field, which reduces heat generation from photothermal conversion and promotes liquid convection, resulting in lower droplet temperatures compared to those under static rising conditions.

39018413_p6

39018413

Motion control of individual droplets

biomedical

Study

en

Furthermore, by decreasing the light intensity and directing it onto one side of the droplet, we can induce controlled 2D motion of the droplet. This reduction in light intensity prevents the expansion of bubbles inside the droplet from generating enough force to counteract gravity, inhibiting upward floating motion. Simultaneously, irradiation on one side of the droplet governs the direction of the Marangoni flow, thereby dictating the droplet’s motion. When the light field is illuminated at one end of the ferrofluid droplet, the induced internal Marangoni flow can propel the droplet to move horizontally within the horizontal plane . In addition, as demonstrated in fig. S11, the light field can selectively drive one of three ferrofluid droplets to move or manipulate all three droplets to fuse (movie S5). The speed of motion of the ferrofluid droplet in the 2D plane is correlated with the power of the external light field, with higher power resulting in increased motion speed . Overall, the light field can govern the movement of droplets along predetermined trajectories, including traversing slopes . Furthermore, the combined driving of optical and magnetic fields can confer more properties upon the droplets. For instance, controlled splitting of droplets can be achieved initially with a magnetic field, followed by selectively driving sub-droplets toward different target positions using the optical field .

39018413_p7

39018413

Motion control of individual droplets

biomedical

Study

en

We further investigate the behavior of splitting and assembling of the droplets. The motion of an incompressible, immiscible ferrofluid droplet in an incompressible, immiscible medium under the effect of a uniform magnetic field is governed by the following continuity and momentum equations ( 31 , 52 – 54 ): ∇ · u = 0 (1) ρ Du Dt = − ∇ p + ∇ · τ + F σ + F m (2) where Du Dt represents the total derivative of the velocity field, u . The right-hand side of the equation represents the force terms due to pressure, viscosity, surface tension ( F σ ) and magnetic field ( F m ) respectively. When subjected to a uniform oscillating magnetic field, the droplet undergoes splitting. To analyze this phenomenon effectively, we introduce dimensionless groups to reduce the number of variables and identify which dimensionless groups have the most substantial impact on droplet dynamics. The dimensionless groups are defined as ( 53 , 54 ): Re = ρ c R 0 2 γ · η c (3) Ca = η c R 0 γ · σ (4) Bo m = R 0 μ 0 H 0 2 2 σ (5) where Re , Ca , and Bo m represent the Reynolds, capillary, and magnetic bond numbers, respectively. As shown in Fig. 4A , the COMSOL simulation reproduces the splitting process of the droplet. The distribution of the flow field during droplet splitting is shown in fig. S15. In addition, we have investigated the dynamic behavior of droplets at different oscillating frequencies .

39018413_p8

39018413

Fission and assembly mechanism of droplet collectives

biomedical

Study

en

The split sub-droplets can be assembled into collective structures with different modes under different magnetic fields settings. The assembly behavior of droplets is mainly dominated by magnetic and fluid forces, which can be expressed as follows ( 31 ): ∑ α = 1 , β ≠ α N { F α , β m + F α , β t + F α , β r } + F n = F d = 6 πη a ( v − v 0 ) (6) where F α , β m is the magnetic dipole force, F α , β t is the hydrodynamic thrust force, F α , β r is the repulsive force, F n is the net force of gravity, and F d is the drag force. The droplet assembly process under different oscillating magnetic field parameters is reproduced by simulation . The oscillating magnetic field is defined as: B v = [ A O sin ( 2 π ft ) cos θ + C O sin θ 0 A O sin ( 2 π ft ) sin θ + C O cos θ ] (7) where A O is the amplitude of the sinusoidal signal, C O is the intensity of the constant component, and θ is the direction angle. The amplitude ratio (λ = A O / C O ) is proposed. When the parameter λ is 1, the sub-droplets generated after splitting will assemble to form a chain-like structure; when λ is 2, the sub-droplets generated by splitting will assemble to form a layer-stacked structure; and when λ is 3, the sub-droplets generated by splitting will assemble to form an independent column-like structure. During the assembly process, the motion trajectories of sub-droplets inside layer-stacked structure are shown in Fig. 4 (C and D) . The trajectories of the particles indicate that there is positional interchange behavior between the particles during the assembly process, and the spacing gradually becomes smaller along the X direction and larger along the Y direction, finally reaching a stable equilibrium state. The particle trajectory for the cases of λ = 1 and λ = 3 are shown in figs. S17 and S18, respectively.

39018413_p9

39018413

Fission and assembly mechanism of droplet collectives

biomedical

Study

en

Droplets exhibit the ability to split and subsequently self-assemble under a predefined magnetic field into collective formations, manifesting in various modes such as chains, layer-by-layer assemblies, and columns. However, these droplet collectives typically rely on the substrate for assembly and movement under a separate magnetic field ( 31 , 35 , 36 ). The introduction of an optical field provides the droplet collectives with greater degrees of freedom, enhancing their capabilities. Like individual magnetic fluid droplets, the formed collectives can achieve levitation behavior. For instance, in Fig. 5A , under an oscillating magnetic field (with λ = 1), the sub-droplets form chain-like collectives (movie S6). Under side light field irradiation, the entire chain-like collectives can remain stable without further splitting and float upward in motion. Upon turning off the light field, the chain-like collectives descend. Similarly, by adjusting the magnetic field, droplet collectives can form a layered structure (with λ = 2), and these layer-assembled droplet collectives can be suspended under side light field irradiation . Simulation results depict the entire uplift of the droplet collectives and the trajectories of the particles . Furthermore, in Fig. 5C , when driven by a separate magnetic field, ferrofluid droplets can first split and then assemble into a columnar structure (with λ = 3). The maximum height of the columnar structure is limited to four layers of the sub-droplet thickness h 1 (movie S7). This limitation arises because of the magnetic repulsion force preventing further assembly of the columnar structure. Subsequently, a light field is applied to induce translational movement of the basic columnar structure without interfering with other columns. The irradiated column then assembles and eventually reaches a height of h 2 , which is 2.5 times higher than h 1 . In addition, dispersed sub-droplets can be individually manipulated by the light field and assembled into desired patterns, such as triangles and the word “Ferrofluid” .

39018413_p10

39018413

Manipulation of droplet collectives

biomedical

Study

en

While droplet robots propelled by a 3D magnetic field face limitations in selectively driving multiple droplet robots within the same space due to the global nature of the magnetic field itself, as previously discussed ( 46 , 47 ), the proposed bimodal actuation strategy overcomes this challenge. Illustrated in Fig. 6A , this strategy facilitates the sequential splitting of droplets into two sub-droplets through cooperative action of magnetic and light fields, enabling object delivery tasks in both 2D and 3D spaces (movie S8). Initially positioned at the start of an artificially constructed labyrinth at t = 0.0 s, the droplet traverses to the intermediate region driven by a magnetic field within 40.6 s. Subsequently, another oscillating magnetic field is applied to induce controlled splitting of the droplet into two sub-droplets. These sub-droplets are then individually driven by a light field to traverse a vertical wall and reach the target position at t = 180.0 s. Notably, the droplet on the right, guided by the vertical light field, transports a flake to the surface at t = 240.0 s, while the droplet on the left, influenced by a magnetic field, carries a spherical object from one end to the other. The bimodal driving strategy significantly enhances the droplet robot’s versatility, expanding its functionality by orchestrating multiple droplets in tandem to accomplish diverse tasks sequentially.

39018413_p11

39018413

Potential application demonstration of the droplet

biomedical

Study

en

In addition to functioning autonomously and accomplishing various tasks as a liquid robot, droplets can also serve as actuators when combined with other components to assemble a soft robot. Leveraging the remarkable photothermal conversion ability of ferrofluid droplets, they can generate thermobuoyant flow under a light field, enabling 3D motion not only for a single droplet but also for the entire droplet assembly. As depicted in Fig. 6B , when assembled with a hydrogel, a jellyfish-shaped robot can be formed, capable of floating motion under NIR laser irradiation (movie S9). Initially positioned on the substrate at t = 0.0 s, the jellyfish robot rises vertically upward upon vertical light field irradiation, reaching the water surface at t = 126.2 s. Subsequently, driven by the light field, the jellyfish robot moves along the water surface at t = 192.9 s. Moreover, swarm motion of multiple jellyfish robots can be achieved by applying a horizontal light field, as illustrated in Fig. 6C . Under the illumination of a horizontal light field, three jellyfish robots rise sequentially to form a robot swarm (movie S9). Droplets exhibit excellent photothermal conversion efficiency, enabling the powering of jellyfish robot assembly with sunlight to facilitate 3D motion. As illustrated in Fig. 6D , sunlight is focused and vertically irradiated on the jellyfish robots using a magnifying glass, inducing the generation of fluids that cause them to float vertically (movie S9). By sequentially irradiating different jellyfish robots, all of them can achieve upward floating motion. The unique property of fluid droplets to assemble with other droplets or materials such as hydrogels substantially expands the capabilities of soft robots and greatly enlarges their design space.

39018413_p12

39018413

Potential application demonstration of the droplet

biomedical

Study

en

In this study, we introduce a dual-modal driving strategy that synergistically integrates magnetic and optical fields to enable precise 3D motion and manipulation of individual ferrofluidic droplets as well as groups of droplets. These droplets, composed of an oil phase and iron tetraoxide, exhibit exceptional magnetic responsiveness and photothermal efficiency, rendering them highly conducive to controlled manipulation. The optical field serves as a key driver in inducing Marangoni flows around the droplets and triggering volume expansion, thereby enhancing their buoyancy. This combined effect propels both individual droplets and droplet collectives to move in 3D or regulate the morphology of droplet assemblies. In addition, the introduction of magnetic fields enables controlled splitting and assembly of droplets. By fine-tuning the magnetic field parameters, droplets can be precisely divided into multiple sub-droplets on demand. Subsequently, different droplets can be selectively driven by the light field to execute distinct tasks. Furthermore, magnetic field–driven droplets have the capacity to assemble into chain-like or layer-stacked structures, while the application of a light field confers upon these droplet collectives the ability to maneuver in 3D. The mechanism governing dual field–regulated droplet motion is elucidated and validated through comprehensive simulations, corroborating experimental observations. Even in various extreme environments, such as acidic and alkaline liquids, flowing liquids, different biofluids, and sticky glycerin, our proposed droplet 3D manipulation method is still feasible . These findings present an efficient strategy for droplet manipulation, broadening the capabilities of droplet-based robotics.

39018413_p13

39018413

DISCUSSION

biomedical

Study

en

A notable challenge in magnetic control is the selective control of multiple untethered robots. Different from traditional robots, it is difficult to equip miniature soft robots with actuators and onboard sensors for motion and control. Therefore, independent control of multiple miniature robots is challenging as all magnetic robots receive the same control inputs in a single external field ( 55 – 57 ). Previously unidentified actuation strategies are desired to achieve selective control and expand the operating efficiency of magnetic robots. In the current work, we propose a feasible solution to combine the global magnetic field with the optical field, which has an adjustable operation region. We demonstrate the utilization of 3D moving droplets as droplet robots for cargo transportation or their integration with other modules to form soft robots capable of controlled movements driven by light fields. This approach represents a solid advancement in droplet manipulation, offering exciting prospects for the development of dynamic and adaptive miniature systems. Furthermore, within different formats of microrobots, liquid microrobots, particularly those structured as droplets, exhibit noteworthy attributes. Their fluidic composition endows them with an exceptional capacity to maneuver through constrictions significantly smaller than their original dimensions, thereby exemplifying a remarkable versatility in locomotion. In addition, the inherent fluidity of droplet microrobots offers substantial operational flexibility, facilitating actions such as the partitioning of a single droplet into multiple sub-droplets or the fusion of disparate sub-droplets into a unified entity. Such capabilities render droplet microrobots proficient in tasks ranging from cargo conveyance to micro-assembly, an advantageous trait particularly esteemed in the biochemical applications.

39018413_p14

39018413

DISCUSSION

biomedical

Study

en

The droplet manipulation methods proposed in this article have several limitations. First, the application of droplet 3D manipulation may be limited because of the limited penetration depth of the light field. Using a catheter to introduce the light source into deeper opaque regions is a viable strategy to extend optical actuation to more fields. Second, strong light fields induce intense temperature changes when driving droplets moving in three dimensions, which are unfavorable for applications such as bio-sample transportation and temperature-sensitive reactions. However, the temperature rise may be exploited for hyperthermia therapy. To avoid drastic temperature rises that could cause harm to the human body or biological samples, low-power density light fields should be used in these applications. Another potential improvement is to increase the initial bubble volume and decrease the density of the droplets so that they can lift under lower temperatures.

39018413_p15

39018413

DISCUSSION

biomedical

Study

en

The fabrication process of oleic acid–coated Fe 3 O 4 nanoparticles involved several steps. Initially, solutions of FeCl 3 (1.2 g/ml) and FeCl 2 (1.3 g/ml) were prepared, mixed in a beaker, and mechanically stirred for 20 min. The solution was then heated in an 80°C water bath. Concentrated ammonia was subsequently added dropwise until the pH reached 10, followed by continued reaction for 35 min. Next, a mixture of 4 g of oleic acid and 20 ml of ammonia was added to the reaction beaker and stirred for an additional 40 min. Dilute hydrochloric acid was then added dropwise until the pH was adjusted to 7. After allowing the solution to precipitate completely, it was discarded, and the precipitate was washed three to five times with anhydrous ethanol. The resulting precipitate was dried in a vacuum oven, ground, and stored in brown glass bottles. This powder was further washed with anhydrous ethanol, dried, mixed with an appropriate amount of corn oil, ground, and stirred. After standing for 24 hours, the supernatant obtained constituted the desired oil-based ferrofluid. Notably, the ferrofluids we produce are not indispensable for the optical response; we have conducted tests on commercial ferrofluids (EMG 905, FerroTec), which also exhibit behaviors akin to ours . This similarity arises from the fact that the optical response of the droplets primarily hinges on their main constituent, iron tetraoxide nanoparticles, which have light-absorbing properties, particularly in the NIR spectrum, and convert this absorbed energy into thermal energy. To enhance optical responsiveness, it is preferable to select a driving light source with wavelengths typically falling between 790 and 2526 nm.

39018413_p16

39018413

Preparation of the ferrofluids and the hydrogel-ferrofluid composites

biomedical

Study

en

To build the jellyfish-like robots, hydrogel-ferrofluid composites were fabricated via a mold-casting method . First, molds A and B were prepared with 3D-printed polylactide material. A hydrogel precursor comprising acrylamide, N , N ′-methylenebisacrylamide, ammonium persulfate, N , N , N ′, N ′-tetramethylethane-1,2-diamine, and deionized water (10:0.1:0.1:0.01:89.79 in wt%, chemicals purchased from Sigma-Aldrich without any modification) was injected into the dome-shaped cavity between mold A and B. After curing, the dome-shaped hydrogel was carefully demolded and put into mold C leaving the opening toward the outside. Then, 10 μl of ferrofluid was injected into the cavity of the dome-like hydrogel, and another hydrogel precursor was injected into the mold to seal the ferrofluid droplet in the hydrogel. During injection, a magnet is used to keep the location of the ferrofluid droplet.

39018413_p17

39018413

Preparation of the ferrofluids and the hydrogel-ferrofluid composites

biomedical

Study

en

A setup comprising three orthogonal pairs of custom-made electromagnets was used, with an internal chamber size of 50 mm by 50 mm by 50 mm. Software-controlled signals dictated the input currents driving the electromagnets through a custom electronic board, enabling adjustable magnetic field intensities ranging from off to a maximum of 9 mT. The light source used in the study was an 808-nm NIR light from Hi-Tech Optoelectronics , offering adjustable optical field power levels from 0 to 15 W. A 3D printer (Pro2, RAISE3D) was used to construct various terrains, including gaps, walls, and channels, used in the experimental setup. High-speed camera observations were conducted using an M310 camera from Phantom Inc. to analyze the motion behavior of the droplets.

39018413_p18

39018413

Setup for experiments

biomedical

Study

en

The temperature of the ferrofluid droplet under varying light field intensities was measured using an infrared camera (ETS320, Teledyne FLIR). To ensure accurate positioning, the ferrofluid droplet was visually centered in the tank at the desired height, with permanent magnets placed beneath the tank to stabilize the droplets. Given the fixed focus distance of 7 cm for the infrared camera, it was positioned 7 cm above the ferrofluid droplet during the experiments. Before testing, the camera underwent calibration using reference points: melting ice and boiling water. For the experiments, 10 μl of ferrofluid was meticulously introduced into the same container under both empty and water-filled conditions. Subsequently, the temperature variations of the ferrofluid droplets, subjected to continuous light field input, were recorded and analyzed.

39018413_p19

39018413

Thermal characterization of the ferrofluid droplets

biomedical

Study

en

Figure S29 illustrates a schematic representation of a ferrofluid droplet suspended in another fluid medium experiencing an oscillating magnetic field, B v ( 7 ). In this scenario, the magnetic susceptibility of the ferrofluid droplet was assumed to be 2.65 (χ d ), while it was considered zero (χ c ) for the suspending nonmagnetic fluid. Subscripts c and d denote the continuous phase and droplet, respectively. The viscosity and density of both phases were assumed to be η c = 6 cP, η d = 1 cP, and ρ d = 1290 kg/m 3 , ρ c = 1000 kg/m 3 individually. The dimensions of the computational domain were set to H domain = W domain = 16 R 0 , where R 0 represents the radius of the undeformed ferrofluid droplet. Initially, the ferrofluid droplet is positioned at the center of the computational domain. The surface tension of ferrofluid is considered as 13.5 mN/m, and surface tension of water is considered as 73 mN/m. This setup investigates the deformation of the droplet under the oscillating magnetic field.

39018413_p20

39018413

Simulation of droplet splitting

biomedical

Study

en

A two-phase laminar flow model using the level set method, combined with transient simulation and phase initialization, was used to simulate the flow domain and track the deformable interface of the droplet. The level set function, denoted as Φ, is assigned values of 1 and 0 for the droplet phase and continuous phase, respectively, to define the interface of the droplet using initial interface conditions. The reinitialization parameter, γ was set equal to the maximum magnitude of the velocity in the flow domain, and the interface thickness, ε was chosen to be of the same order as the size of the mesh elements. In addition, a magnetic field was applied to the flow domain and solved simultaneously using the magnetic fields and no currents interface from AC/DC module. In this case, the magnetic field was applied in 2D plane, and magnetic insulation was applied to both left and right walls. The time-dependent solver is used for analyzing droplet deformation under the varying magnetic field frequency 0.5 to 10 Hz. The mesh was created using free triangular elements in the computational domain, and the PARDISO solver with nested dissection multithread algorithm is used to solve the computational model.

39018413_p21

39018413

Simulation of droplet splitting

biomedical

Study

en

A two-phase laminar flow model using the level set method, combined with transient simulation and phase initialization, was used to simulate the flow domain. A Marangoni effect module was applied to the boundary of two phases to couple the flow field with heat transfer, where the surface tension coefficient was set as default from the materials library. A heat source was applied to the ferrofluid phase with equation q 0 = 10 8* Φ[ Wm −3 ], where Φ = 1 for the ferrofluid phase and Φ = 0 for the nonmagnetic phase. Droplet splitting and floating were simulated by COMSOL Multiphysics software.

39018413_p22

39018413

Simulation of droplet floating due to light irradiation

biomedical

Study

en

On the basis of Eq. 6 , we conducted simulations to investigate the motion behavior of colloidal particles. Our custom interface programmed with MATLAB software was used to study the assembling process of droplet collectives under the influence of an oscillating magnetic field. In our simulations, the droplet diameter was fixed at 1.5 mm, the colloid density was set to 1290 kg/m 3 , and the magnetic susceptibility was set as 2.65. The strength of magnetic field was set as 9 mT, and the frequency was varied from 1 to 100 Hz. The parameters of droplet in the simulation are summarized in table S1.

39018413_p23

39018413

Simulation of droplet assembling

biomedical

Study

en

Rice aroma is generated by the interaction between volatiles in rice and olfactory receptors. It's one of the vital attributes that influenced the popularity of rice, and affected consumer preference to a certain extent . Therefore, aromatic rice with good appearance, texture and fragrance is more popular with consumer in the market, and more expensive than non-aromatic rice.

39021608_p0

39021608

introduction

other

Other

en

Currently, more than 500 volatile compounds have been detected in aromatic and non-aromatic rice, including aldehydes, ketones, alcohols, phenols, esters and heterocyclics and other compounds. Although many volatiles had been identified, only a few of them were considered to have important contributions to rice aroma . The characteristic volatiles in rice samples were investigated in many works . Since the 20th century, 2-acetyl-1-pyrroline (2-AP) has been reported to be a key aroma compound in rice in multiple studies, providing the flavor of popcorn . It was considered as the most important discriminator between aromatic and non-aromatic rice. However, rice samples with similar 2-AP content might have different aroma quality, suggesting that some volatiles other than 2-AP also had important contribution to rice aroma. And different characteristic volatiles were obtained for different rice samples.

39021608_p1

39021608

introduction

biomedical

Study

en

Heptanal, octanal, trans -2-decenal, 1-heptanol, trans -2-decen-1-ol, 3,7,11-trimethyl-3-dodecanol, 3-octene-2-one, and 2-AP were considered as biomarkers for distinguishing Wuchang rice from other rice . Zhao et al. considered 22 volatile compounds (including benzaldehyde, 2-pentylfuran, trans -2-nonenal, 3-octen-2-one, 1-octanol, nonanal, 2-methoxy-4-vinylphenol, trans -2-heptenal, 2-octen-1-ol and so on) as key volatiles in cooked rice form different regions in China. 1-Octen-3-ol, 1-ethyl-3,5-dimethylbenzene, 2,6,11-trimethyldodecane, 3-ethyloctane, 2,7,10-trimethyldodecane, methyl salicylate, 2-octanone, and heptanal were selected as important compounds to discriminate different japonica rice cultivars . However, there was no conclusion as to which volatiles played a key role in the overall aroma of rice and could be used as key aroma compounds for evaluating rice aroma, making it difficult to make a breakthrough in the method of evaluating rice aroma quality.

39021608_p2

39021608

introduction

biomedical

Study

en

Meanwhile, the aroma system of rice is very complex and not all volatile compounds have positive effects on rice aroma. Some compounds such as α -pyrrolidone, pyridine, guaiacol, indole and p -xylene were reported to possess fruity and floral odors and be beneficial to rice aroma, but lipid oxidation products such as hexanal, trans -2-octenal, octanal, and decanal were reported to possess undesirable odors and have negatively effect on rice aroma . Therefore, the evaluation of rice aroma needs to be combined with other volatile compounds rather than using by 2-AP alone to evaluate rice aroma.

39021608_p3

39021608

introduction

other

Other

en

Gas chromatography-mass spectrometry (GC-MS) was widely used for qualitive and quantitative volatile compounds in rice. Since GC-MS can't directly explain the aroma of volatile compounds, it is often used in combination with gas chromatography-olfactometry (GC-O) and odor activity value (OAV) to evaluate the importance of volatile compounds to the overall flavor. However, the interactions between different volatile compounds would influence the final perceived. High levels of 1-propanol and 2-phenylethanol were reported to significantly inhibit the volatilization of 3-methylbutyric acid from liquor . In cheese, δ -dodecalactone promoted the expression of lactone fruity flavor, but γ -dodecalactone had an inhibitory effect on the expression of lactone fruity flavor . GC-O and OAV analysis ignore the interaction between volatile compounds. Hence, the GC-O and OVA results need further validation.

39021608_p4

39021608

introduction

biomedical

Study

en

In this paper, multiple analysis techniques including GC-MS, GC-O, OAV analysis and sensory analysis were applied to analyze the characteristic volatiles in rice and their influence on cooked rice aroma. The volatiles in rice were first analyzed and quantified by using GC-MS. Then, GC-O analysis, correlation analysis between sensory scores and volatile contents, and OAV analysis were carried out to screen the main potential characteristic volatiles. Finally, the effects of the potential characteristic volatiles on the aroma quality of rice were investigated by sensory methods including sensory ranking and triangle test.

39021608_p5

39021608

introduction

biomedical

Study

en

Thirty-one rice varieties (Suyunuo, Daohuaxiang, Meixiangzhan, Yuzhenxiang, Della, Basmati 370, Xiangjingnuo, XiangjingR109, Suxiangjing1hao, Xiangjing 111, Baimaoxiangnuo, Kajinuo, Zhongxiang1hao, Wuxiangjing 14, Dahuaxiangnuo, Yixiang B, Luxiang 90, Songxiang 06–317, Longxiang 04, Wuyou A, Chuanxiang 29B, Longfeng 06, Nongxiang 99, Yuzhuxiang, Meiguoxiangdao, Jasmine 85, Zhongjia 17, Zhonghua 11, Koshihikari, Zhong 2B, D50) were harvested in 2021 and 2022. Thirty-one samples harvested in 2021 were used for volatile profile analysis by GC-MS. Nine of the 31 samples planted and harvested in 2022 were used for GC-O analysis. After dehulled by a sheller (Satake, Tokyo, Japan) and milled by rice a polisher , the rice samples were stored at the temperature of 4 °C, and analyzed within half a month.

39021608_p6

39021608

samples and chemicals

biomedical

Study

en

2-Methyl-3-heptanedone used as internal standards, 2-pentylfuran, octanal, trans -2-octenal, 1-octen-3-ol, decanal and trans -2-nonenal were purchased from Tokyo Chemical Industry (Shanghai, China). Hexanal, isopropanol and trans , trans -2.4-decadienal were obtained from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China), and 2-AP (10% w/w in toluene) was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

39021608_p7

39021608

samples and chemicals

biomedical

Other

en

The rice sample was cooked according the method in Chinese Agricultural Industry Standard NY/T 3837-2021 with some modifications. Briefly, 30g of milled rice was weighed into an aluminum box and washed with deionized water for twice. After adding appropriate deionized water (30 g for glutinous rice, 37.5 g for non-glutinous rice), the sample was sealed and soaked for 30 min. Then, the rice sample was steamed for 40min and simmered for 20min, and ready for the following analysis.

39021608_p8

39021608

preparation of cooked rice

biomedical

Other

en

After 5g of cooked rice and 10 μL of 1 μg/mL 2-methyl-3-heptanone were added into a 40 mL brown extraction vial, the vial was sealed. The solid phase microextraction (SPME) fiber ((DVB/CAR/PDMS, 50/30 μm, 1 cm), Anpel, Shanghai, China) was exposed to the headspace of the vial at a temperature of 80 °C for 30 min. Then, the SPME fiber was inserted into the injection port of GC-MS , and desorbed at 250 °C for 5 min. Spilt mode (5:1) was applied during injection. The volatiles were separated and evaluated by using a DB-WAX column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies Co.) with high-purity helium (purity >99.999%) as carrier gas at a flow rate of 1 mL/min. The oven temperature was set as 40 °C for 5 min, then programmed to 230 °C at 5 °C/min and maintained for 10 min. The mass selective detector was operated in electronic impact ionization mode (70 eV) with a scan range of m/z 40–500. The ion source temperature was 230 °C. All experiments were performed in triplicate.

39021608_p9

39021608

gas chromatography-mass spectrometry analysis

biomedical

Study

en

The volatiles were identified first by comparing the mass spectra with those in the NIST 14 spectral database and self-established rice volatile compounds database, and then by comparing the Kovates’ retention indices (RIs) calculated from the retention times of a series of n-alkanes (C6–C24) (Equation 1) with reference values provided by NIST14. The relative content of volatiles were calculated by using Equation 2. (1) RI ( X ) = 100 Z + 100 [ RT R ( X ) − RT R ( Z ) ] / [ RT R ( Z + 1 ) − RT R ( Z ) ] Where RTR(X), RTR(Z), RTR(Z+1) represent the retention time of tested compound x and n-alkanes with carbon numbers of Z, Z+1, respectively, and RTR(Z) < RTR(X) < RTR(Z+1). (2) C = A 1 A × cv m where C is the relative content of tested volatile (ng/g); c is the concentration of 2-methyl-3-heptanone (μg/mL); A and A1 are the peak area of 2-methyl-3-heptanone and tested volatiles, respectively; v is volume of 2-methyl-3-heptanone (μL); m is mass of cooked rice (g).

39021608_p10

39021608

gas chromatography-mass spectrometry analysis

biomedical

Study

en

An olfactory detector was coupled to GC for the identification of odor-active compounds. The extraction procedure and instrument conditions for GC were basically the same as those described in section 2.3 , except that the split mode was set to 2.5:1. Sensory panelists sniffed and recorded the odor characteristics, intensity and duration of the stimuli as well as their retention time. A 5-point scale was used for intensity ratings. All experiments were repeated five times.

39021608_p11

39021608

gas chromatography-olfactometry analysis

biomedical

Study

en

The sensory analysis was carried out in the sensory laboratory of Rice Product Quality Supervision and Inspection Centre, Ministry of Agriculture and Rural Affairs. Twelve sensory panelists (5 males and 7 females) were selected from the sensory laboratory of Rice Product Quality Supervision and Inspection Centre, Ministry of Agriculture and Rural Affairs, according to the GB/T 16291.1-2012 (Chinese National Standard). One week prior to the sensory experiment, the sensory panelists were trained once a day for half an hour on the purpose and methodology of the experiment, including the knowledge and description of the samples. During sensory analysis, at least 1 min was allowed to elapse between the evaluation of two samples, and no more than 7 samples were evaluated at one time, in order to avoid a "carry-over" effect.

39021608_p12

39021608

sensory analysis

other

Study

en

Each panelist was authorized to conduct sensory analysis, had at least three years of sensory experience and had participated in sensory evaluation tests for rice flavor and eating quality. All samples used in the sensory analysis were non-toxic and no side effects on the body. And the sensory panelists in this study gave informed consent via the statement “I am aware that my responses are confidential, and I agree to participate in this study” where an affirmative reply was required to enter the study. They can withdraw from the study at any time without any reason.

39021608_p13

39021608

sensory analysis

biomedical

Study

en

The sensory score evaluation of cooked rice was performed according to NY/T 596–2002 (Chinese Agricultural Industry Standard). The rice sample was first cooked as mentioned in section 2.2 and then scored by five sensory panelists with respect to the intensity of rice popcorn aroma. Very strong: 9–10 points; strong: 7–8 points; medium: 5–6 points; weak: 3–4 points; recognized or around the threshold: 1–2 points; no popcorn aroma: 0 point. The average score of 5 panelists was used as the final sensory score of rice aroma.

39021608_p14

39021608

sensory score evaluation

other

Other

en

The sensory ranking was performed with reference to GB/T 12318-2008 (Chinese National Standard). In order to simulate the aroma of volatiles in rice, five volatile solutions (10 μL, in isopropanol solution) with five concentrations were added to aluminum boxes, respectively. The simulated contents covered the contents of test volatiles in rice samples. After sealed and randomly numbered, the aluminum boxes were ranked by seven panelists according to odor intensity and preference level. The concentration of standard solution added was calculated by using Equation (3) . Since relative contents of volatiles were obtained in section 2.3 , correction factors (f) were determined by using the method in Chinese Light Industry standards , and facilitated the calculation of volatile contents. (3) C 1 = m 1 × C × f / v 1 Where C 1 is the content of volatile in standard solutions (μg/mL) and C is the relative content of volatile in rice (ng/g); f is the correction factor; m 1 is mass of cooked rice (g); v 1 is volume of standard solution added (μL). The volatile contents in standard solutions and corresponding simulated contents in the rice samples were shown in Table S1 .

39021608_p15

39021608

sensory ranking

biomedical

Study

en

To assess whether there were significant differences between samples, F test was determined according to Equation 4. There were significant differences among samples ( p ≤ 0.05) if F test > F (9.11); otherwise, there were no significant differences. In order to explore which samples were significantly different from others, the least significant difference (LSD) was further calculated by using Equation 5. There were significant differences between two samples ( p ≤ 0.05) if the difference in rank between two samples was equal to or greater than LSD; otherwise, there was no significant difference. (4) F test = 12 / jp ( p + 1 ) ( R 1 2 + … + R i 2 ) − 3 j ( p + 1 ) (5) LSD = z jp ( p + 1 ) 6 where j and p are the numbers of panelists and samples, respectively; R i is the rank sum of the ith sample and z value is 1.96 ( p ≤ 0.05).

39021608_p16

39021608

sensory ranking

biomedical

Study

en

Triangle test was carried out according to ISO 4120-2021. During the test, panelists was given a set of three cooked rice samples and informed that two of the samples were the same and the other was different. The set of rice samples contained the same cooked rice and a standard solution of one volatile had been added to one or two of the samples. Panelists reported which sample they thought was different and described the aroma differences. The test was repeated 24 times for each volatile. There was a perceptible difference between the samples with and without adding volatile, if the number of correct responses was greater than or equal to the number given in the standard (13/24, p ≤ 0.05).

39021608_p17

39021608

triangle test

biomedical

Study

en

Eighty-five volatile compounds were detected by GC-MS in rice samples ( Table 1 ), including 2-AP, acids (3), alcohols (11), aldehydes (17), alkanes (4), aromatics (12), esters (7), furans (5), ketones (16) and others (9). Among the volatiles, only 4 volatiles were detected in all rice samples, namely hexanal, 2-pentylfuran, nonanal and trans -2-octenal. Aldehydes were the most abundant in rice, accounting for 37.28–86.82% of total volatiles . Among the aldehydes, nonanal (4.52–106.92 ng/g) and hexanal (1.63–48.38 ng/g) were more abundant than other aldehydes ( Table 1 ). In addition, a high relative content of furans (3.63–21.26%) was obtained. And 2-pentylfuran was the most abundant furan, whose relative content was 2.54–30.31 ng/g. It was considered as one of the important compounds to distinguish aromatic rice from non-aromatic rice . The proportions of ketones and alcohols ranged from 2.32 to 16.35% and 2.37–11.22%, respectively. Among the ketones and alcohols, 3-nonen-2-one, 6-methyl-5-hepten-2-one and 1-octen-3-ol played an important role in rice aroma due to their high contents and low odor thresholds. However, esters, acids and alkanes had low relative content proportions, accounting for 0–14.52%, 0–3.86% and 0–1.33%, respectively. In that they had high thresholds, only few esters, acids and alkanes produced unique aromas in rice. Their contribution to rice aroma was limited. The content of 2-AP in test rice samples were 0–5.18 ng/g, accounting for 0–7.48% of total volatiles. Owing to the low threshold (0.053 ng/g), 2-AP made an important contribution to rice aroma and was used to distinguish rice varieties . Besides, rice also contained a large number of other volatiles, such as indole, 4-ethylphenol, pyridine and p-cymene etc., which accounted for 0–49.92% of total volatiles. Among these, indole possessed the highest content (0–47.75 ng/g). It was reported to be one of the characteristic volatiles of the unique aroma of black rice, was higher in freshly cooked rice and decreased slightly with prolonged storage . Fig. 1 The profile of chemical group proportion of volatiles in rice samples. Fig. 1 Table 1 The relative contents of volatiles in rice samples. Table 1 NO. Classified Compounds Odor a Frequency b Threshold c (ng/g) Content (ng/g) OAV RI d Comp1 Aldehydes Hexanal green tomato, green and grass-like 31 5 1.63-48.38 0.33-9.68 1071/1078 Comp2 Aldehydes Heptanal fruity, fatty and rancid-like 28 2.8 0–3.69 0–1.32 1174/1185 Comp3 Aldehydes Octanal citrus, fruity, floral, and fatty 25 0 0.587 0–16.62 0–28.31 1277/1286 Comp4 Aldehydes trans -2-Heptenal fruity, green, fatty 30 40 0–4.84 <1 1308/1334 Comp5 Aldehydes Nonanal fat, citrus, green 31 1.1 4.52–106.92 4.11–97.20 1381/1395 Comp6 Aldehydes trans -2-Octenal green and fatty-like 31 3 1.04–7.32 0.35-2.44 1414/1428 Comp7 Aldehydes trans , trans -2,4-Heptadienal fatty, sweet, fruity citrus 13 15.4 0–0.46 <1 1479/1490 Comp8 Aldehydes Decanal soap, orange peel, tallow 17 3 0–22.06 0–7.35 1482/1500 Comp9 Aldehydes Benzaldehyde nutty and bitter-like 29 750.89 0–8.10 <1 1504/1508 Comp10 Aldehydes trans -2-Nonenal fatty, tallow, beany, cucumber and woody-like 30 0.19 0–5.96 0–31.37 1523/1532 Comp11 Aldehydes Benzeneacetaldehyde floral, herbal 6 6.3 0–0.76 <1 1619/1636 Comp12 Aldehydes trans -2-Decenal fatty and waxy-like 24 17–250 0–1.25 <1 1629/1634 Comp13 Aldehydes 2-Butyl-2-octenal green, vegetable, cucumber, fatty 17 20 0–4.86 <1 1655/1659 Comp14 Aldehydes trans , trans - 2,4-Nonadienal fatty, waxy and nutty-like 2 0.1 0–0.57 0–5.7 1681/1686 Comp15 Aldehydes 2-Undecenal sweet 7 – 0–0.90 – 1737/1755 Comp16 Aldehydes trans , trans -2,4-Decadienal chicken, fatty 30 0.077 0–6.83 0–88.70 1793/1805 Comp17 Aldehydes 2,6,6-Trimethyl-1-cyclohexene-1-carboxaldehyde – 2 – 0–0.08 – 1604/1590 Comp18 2-AP 2-Acetyl-1-pyrroline popcorn, sweet 27 0.053 0–5.18 0–97.74 1321/1331 Comp19 Acids Nonanoic acid waxy, dirty 1 4600–9000 0–0.05 <1 2155/2174 Comp20 Acids Tetradecanoic acid – 14 10000 0–1.29 <1 2669/2685 Comp21 Acids n-Hexadecanoic acid waxy, fatty 26 20000 0–5.50 <1 2879/2875 Comp22 Alcohols 1-Octen-3-ol mushroom 30 1.5 0–9.17 0–6.11 1445/1459 Comp23 Alcohols 2-Methyl-6-hepten-1-ol – 1 – 0–0.43 – 1459/1480 Comp24 Alcohols 2-Ethyl-1-hexanol citrus, floral, oily, sweet 2 25482.2 0–1.65 <1 1485/1494 Comp25 Alcohols Linalol lemon; orange; citrus; floral; sweet 3 0.22 0–0.47 0–2.14 1541/1554 Comp26 Alcohols 1-Octanol citrus, fruity and floral-like 2 125.8 0–4.47 <1 1552/1558 Comp27 Alcohols (Z)- 5-Octen-1-ol green, melon, mushroom 7 6 0–1.96 <1 1606/1608 Comp28 Alcohols 1-Nonanol floral and citrus-like 4 45.5 0–0.63 <1 1653/1663 Comp29 Alcohols α, α -Dimethylbenzenemethanol – 6 – 0–0.21 – 1744/1759 Comp30 Alcohols trans -3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol – 3 – 0–0.40 – 2029/2028 Comp31 Alcohols Cedrol cedarwood 28 – 0–2.90 – 2101/2016 Comp32 Alcohols 2-Phenoxyethanol – 5 – 0–0.20 – 2121/2107 Comp33 Alkanes Heptane – 2 5000 0–0.08 <1 704/705 Comp34 Alkanes Tricyclo[2.2.1.0(2,6)]heptane, 1,7-dimethyl-7-(4-methyl-3-pentenyl) – 9 – 0–0.95 – 1560/1555 Comp35 Alkanes β -Copaene – 2 – 0–0.44 – 1579/1562 Comp36 Alkanes Diphenylmethane sweet, green, wet, plastic 2 – 0–0.14 – 1990/1994 Comp37 Aromatics Toluene ethereal-like 29 527 0–0.59 <1 1027/1036 Comp38 Aromatics Ethylbenzene – 6 2205 0–0.12 <1 11112/1123 Comp39 Aromatics 1,3-Dimethylbenzene – 5 – 0–0.22 – 1126/1140 NO. Classified Compounds Odor a Frequency b Threshold c (ng/g) Content (ng/g) OAV RI d Comp40 Aromatics Styrene balsamic, gasoline 29 65 0–1.55 <1 1241/1250 Comp41 Aromatics Naphthalene tar 30 6 0–1.07 <1 1714/1712 Comp42 Aromatics 1,3-Dimethoxybenzene – 1 – 0–0.60 – 1727/1730 Comp43 Aromatics 1,2,3,5,6,8a-Hexahydro-4,7-dimethyl-1-(1-methylethyl)-, (1S-cis)- Naphthalene – 9 6 0–0.28 <1 1742/1759 Comp44 Aromatics (R)-1-Methyl-4-(1,2,2-trimethylcyclopentyl)-benzene – 4 – 0–0.49 – 1802/1825 Comp45 Aromatics Butylated hydroxytoluene phenolic, camphoreous 4 – 0–0.78 – 1897/1911 Comp46 Aromatics 4-Isopropyl-6-methyl-1-methylene-1,2,3,4-tetrahydronaphthalene – 2 – 0–0.03 – 1938/1954 Comp47 Aromatics Biphenyl pungent, green, geranium 3 0. 5 0–0.11 <1 1963/1967 Comp48 Aromatics 1,6-Dimethyl-4-(1-methylethyl)- naphthalene – 30 – 0–0.26 – 2198/2200 Comp49 Esters 1,6-Octadien-3-ol, 3,7-dimethyl-formate – 2 – 0–0.24 – 1541/1579 Comp50 Esters Hexadecanoic acid, methyl ester – 3 – 0–0.14 – 2206/2223 Comp51 Esters Hexadecanoic acid, ethyl ester – 3 – 0–2.40 – 2246/2270 Comp52 Esters Diethyl phthalate – 11 – 0–1.19 – 2350/2359 Comp53 Esters 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester – 26 – 0–6.30 – 2520/2526 NO. Classified Compounds Odor a Frequency b Threshold c (ng/g) Content (ng/g) OAV RI d Comp54 Esters Dibutyl phthalate – 29 – 0–3.56 – 2660/2678 Comp55 Esters Methyl salicylate Wintergreen, minty 7 40 0–0.36 <1 1752/1753 Comp56 Furans 2-Propylfuran – 2 – 0–0.04 – 1023/1011 Comp57 Furans 2-n-Butylfuran nutty, roasted 3 27 – 0–1.79 – 1125/1123 Comp58 Furans 2-Pentylfuran floral, fruity, nutty, bean 31 5.8 2.54-30.31 0.44-5.23 1222/1229 Comp59 Furans 2-Hexylfuran – 1 – 0–0.03 – 1318/1323 Comp60 Furans 2-n-Heptylfuran faint, fruity, sweet, wine-like 1 – 0–0.22 – 1423/1429 Comp61 Ketones 2-Heptanone fruity and floral-like 22 140 0–4.12 <1 1172/1184 Comp62 Ketones 3-Octanone nut 12 1.3 0.0.29 <1 1244/1261 Comp63 Ketones 2-Octanone fruity and floral-like 18 50.2 0–8.34 <1 1274/1287 Comp64 Ketones 6-Methyl-5-hepten-2-one banana-like 30 68 0–3.17 <1 1325/1338 Comp65 Ketones 2-Nonanone fruity and herbaceous-like 12 200 0–0.62 <1 1377/1390 Comp66 Ketones 2-Decanone orange-like floral 7 8.3–41 0–0.90 <1 1485/1495 Comp67 Ketones 3-Nonen-2-one pleasant fruity 14 800 0–0.71 <1 1501/1506 Comp68 Ketones 6-Undecanone – 8 85–410 0–0.43 <1 1519/1527 Comp69 Ketones Isophorone camphoreous, fruity, musty 24 – 0–4.93 – 1574/1577 Comp70 Ketones 6-Methyl-3,5-heptadien-2-one – 2 – 0–1.66 – 1576/1582 Comp71 Ketones 2-Undecanone fruity, fatty 10 5.5 0–0.61 <1 1587/1599 Comp72 Ketones 2-Tridecanone fatty, waxy, mushroom 22 – 0–0.88 – 1798/1814 Comp73 Ketones 2-Pentadecanone fatty, spicy, floral 29 – 0–7.62 – 2009/2023 Comp74 Ketones 1-(2-Hydroxy-5-methylphenyl)- ethanone – 6 – 0–2.28 – 2172/2178 NO. Classified Compounds Odor a Frequency b Threshold c (ng/g) Content (ng/g) OAV RI d Comp75 Ketones Benzophenone – 7 – 0–0.96 – 2449/2457 Comp76 Ketones (E)-6,10-dimethylundeca-5,9-dien-2-one Rose, floral, fruity 30 60 0–5.44 <1 1841/1856 Comp77 Others 2-Methoxyphenol – 1 – 0–0.41 – 1838/1836 Comp78 Others Pyridine Pungent-like 8 2000 0–0.21 <1 1169/1176 Comp79 Others p-Cymene solvent, gasoline, citrus 4 5.01 0–0.05 <1 1255/1272 Comp80 Others 2-Pentylthiophene fruity, slightly fatty, cranberry 21 – 0–0.82 – 1444/1452 Comp81 Others Longifolene Sweet, woody 2 – 0–3.00 – 1552/1565 Comp82 Others γ -Cadinene wood 2 – 0–0.13 – 1743/1745 Comp83 Others α -Calacorene wood 14 – 0–0.14 – 1896/1916 Comp84 Others 4-Ethylphenol smoke, phenolic, creosote 1 21 0–0.17 <1 2156/2174 Comp85 Others Indole mothball, burnt 28 40 0–47.75 0–1.19 2413/2414 Notes. b represent the number of times the compound was detected in 31 rice samples. a odor descriptions were obtained from http://www.thegoodscentscompany.com/ ， https://www.flavornet.org/ and Verma, D. K and Srivastav, P. P. . c odor thresholds were obtained from Gemert, L. J. V. . d before “/”: RIs calculated using n-alkanes C6 to C24 as external standards on a DB-Wax column; after “/”: reference RIs obtained from https://webbook.nist.gov/ .

39021608_p18

39021608

characteristic volatile compounds in rice

biomedical

Study

en

GC-O was used for the analysis of odor characteristic compounds. It could effectively explore active-odor compounds from varieties of volatiles. Nine rice samples were analyzed by GC-O, including 3 glutinous rice (Suyunuo; Xiangjingnuo; kajinuo), 3 japonica rice (Suxiangjing1hao; XiangjingR109; Koshihikari) and 3 indica rice (Chuanxiang29B; Yuzhenxiang; Yixiang B). Koshihikari was a non-aromatic rice, and the others were aromatic rice. Ten volatiles were sniffed through GC-O analysis, including 2-AP, 5 aldehydes (hexanal, octanal, trans -2-nonenal, decanal and tans, trans -2,4-decadienal), one alcohol (1-octen-3-ol) and 3 unknown volatiles (unknown1-3) . Fig. 2 The odor intensity and detection frequency of odor-active volatiles sniffed during GC-O analysis. Y1: Suyunuo; Y2: Xiangjingnuo; Y3: kajinuo; Y4: Suxiangjing1hao; Y5: XiangjingR109; Y6: Koshihikari; Y7: Chuanxiang 29B; Y8: Yuzhenxiang; Y9: Yixiang B. Fig. 2

39021608_p19

39021608

characteristic volatile compounds in rice

biomedical

Study

en

The odors of hexanal, octanal, trans -2-nonenal, decanal and trans , trans -2,4-decadienal were described as grassy and fatty, citrus, fatty and soap, citrus, fatty, respectively. Hexanal was only detected in glutinous rice, with odor intensity of 2.25–2.75 and detection frequency of 4 times. As it was an oxidation product of lipid whose content was higher in glutinous rice than non-glutinous rice , it was easier to be smelled in glutinous rice. Octanal was perceived 5 times in Y6, 4 times in Y3 and Y5, 3 times in Y7, with odor intensity varying from 0 to 3. Decanal, trans -2-nonenal and trans , trans -2,4-decadienal were perceived in all samples. The odor intensity of decanal and trans , trans -2,4-decadienal was 1-3, while that of trans -2-nonenal was 2-4.2. Among them, decanal and trans -2-nonenal were perceived in all repeats in six and seven samples, respectively. Trans , trans -2,4-decadienal was perceived in all repeats in one sample (Y7). Meanwhile, 2-AP was perceived in all samples and the odor intensity was 1.8-5 with a popcorn aroma. It was found that the odor intensity of 2-AP in aromatic rice (4-5) were greater than that in non-aromatic rice (1.8, Y6). It was consisted with previous study that higher odor intensity of 2-AP was obtained for aromatic rice than non-aromatic rice . The odor of 1-octen-3-ol was perceived in 7 samples and described as mushroom flavor with the odor intensity ranged from 1 to 3. It was reported to contribute greatly to rice flavor due to its high content and low threshold . In addition, 3 unknown volatiles, which were described as cooked rice, creamy and soapy, respectively, were also sniffed during GC-O analysis.

39021608_p20

39021608

characteristic volatile compounds in rice

biomedical

Study

en

The rice aroma was produced by comprehensive result of volatiles. The correlations between volatile content and the sensory score were investigated . As seen from Fig. 3 , hexanal (Comp1) had a negative correlation with sensory score (r = -0.58) and positive correlations with trans-2-heptenal (Comp4, r = 0.83), trans-2-octenal (Comp6, r = 0.93), 2-butyl-2-octenal (Comp13, r = 0.8), 1-octen-3-ol (Comp22, r = 0.94), 2-n-butylfuran (Comp57, r = 0.91), 2-pentylfuran (Comp58, r = 0.94)，2-heptanone (Comp61, r = 0.93), 2-decanone (Comp66, r = 0.91), 3-nonen-2-one (Comp67, r = 0.83), 6-undecanone (Comp68, r = 0.9). All of them had a negative correlation with sensory score (r < -0.4), except for 2-buty-2-octenal (r = -0.23). Additionally, benzaldehyde (Comp9), benzeneacetaldehyde (Comp11), trans-2-decenal (Comp12), trans, trans-2,4-nonadienal (Comp14), heptane (Com33), 2-Nonanone (Comp65) and 4-ethylphenol (Comp84) also had a negative correlation with sensory score (r < -0.4), and positively correlated with hexanal (r ≥ 0.35). It revealed that hexanal could be a representative of negative volatiles for rice aroma. Fig. 3 Correlation analysis between volatiles and sensory score. The compound numbers in this were the same as those in Table 1 . Fig. 3

39021608_p21

39021608

characteristic volatile compounds in rice

biomedical

Study

en

Octanal (Comp3) was considered as an early oxidation marker and increased during storage . It had positive correlations with decanal (Comp8, r = 0.56) and trans-2-nonenal (Comp10, r = 0.75). They had low correlations with sensory score (octanal, r = −0.16; decanal, r = 0.19, trans-2-nonenal, r = 0.07). Nevertheless, GC-O analysis showed that they were active-odor compounds in rice and had an influence on rice aroma. Nonanal (Comp5), an abundant volatile in rice, was one of the most important volatiles, contributing to the aroma profile of different rice varieties . It had a positive correlation (r = 0.88) with trans-2-nonenal and also had a low correlation (r = 0.01) with sensory score. Trans, trans-2,4-decadienal (Comp16) had positive correlation (r = 0.46) with 2-AP (Comp18). However, both trans, trans-2,4-decadienal and 2-AP were weakly correlated with sensory score (r = 0.18, −0.02, respectively). Besides, trans, trans-2,4-decadienal, 2-AP also had positive correlations with trans-2-heptenal (Comp4) and p -cymene (Comp79) (r = 0.41, 0.51 respectively). The low correlation between 2-AP and sensory score might be caused by the influence of other volatiles. Correlation analysis was implemented by using rice samples with hexanal, 2-pentylfuran, trans-2-octenal and 1-octen-3-ol content around the odor thresholds, respectively. The results showed that the correlation between 2-AP and sensory score was significantly improved by using rice samples with low contents of hexanal, 2-pentylfuran, trans-2-octenal and 1-octen-3-ol, which increased to 0.38, 0.31, 0.46 and 0.41, respectively. It was indicted that the perception sensitivity of 2-AP was reduced due to the high content of negative volatiles.

39021608_p22

39021608

characteristic volatile compounds in rice

biomedical

Study

en

1-Octen-3-ol was a degradation product of linoleic acid, and was an odor-active alcohol with a mushroom flavor. It was considered as a source of unpleasant odor in rice bran and increased with time during storage . It was positively correlated with hexanal (r = 0.94), trans-2-octenal (r = 0.95), and 2-pentylfuran (r = 0.89). Trans-2-octenal was reported to be associated with the nutty and roasty flavors of rice . 2-Pentylfuran was an important odor active in wild rice , whose flavor was described as bean, green and almond. It was reported that hexanal, trans-2-octanal, 2-pentylfuran, and 1-octen-3-ol were often used as markers of rice ageing in previous studies . It was consistent with the fact that they were negatively correlated with the sensory score. Hexanal, 2-pentylfuran, trans-2-octenal and 1-octen-3-ol were positively correlated (r ≥ 0.89) with each other. Thus, they might have an additive or synergistic effect with each other and adversely contribute to rice aroma. Ketones contributed fruity, nutty, floral flavor to rice aroma. A positive correlation (r = 0.47) was found between 2-pentadecanone (Comp 61) and sensory score.

39021608_p23

39021608

characteristic volatile compounds in rice

biomedical

Study

en

OAV analysis is an important method to evaluate the contribution of volatiles to food aroma. It was implemented by evaluating the ratio of volatile content to odor threshold. In this paper, the relative contents of volatiles were obtained, and the relative OAVs were calculated ( Table 1 ). Fourteen volatiles in rice were found to have relative OAVs greater than 1, namely hexanal, heptanal, 2-pentylfuran, octanal, 2-AP, nonanal, trans-2-octenal, 1-octen-3-ol, decanal, trans-2-nonenal, linalool, trans, trans-2,4-nonadienal, trans, trans-2,4-decadienal and indole ( Table 1 ). Among them, 2-AP, nonanal, trans, trans-2,4-decadienal, trans2-nonenal and octanal had relative OAVs higher than 10, which could reach to 97.74, 97.20, 88.70, 31.37 and 28.31, respectively. Hence, 2-AP, nonanal, trans, trans-2,4-decadienal, trans2-nonenal and octanal were considered to have great contributions to rice aroma due to their high relative OVAs. The relative OAVs of hexanal, heptanal, 2-pentylfuran, trans-2-octenal, 1-octen-3-ol, decanal, linalool, trans, trans-2,4-nonadienal, and indole could reach to 9.68, 1.32, 5.23, 2.44, 6.11, 7.35, 2.14, 5.7 and 1.19, respectively. They were also supposed to contribute considerably to the aroma of rice. Among them, 1-octen-3-ol, hexanal, trans-2-octenal, 2-pentylfuran, and trans, trans-2,4-nonadienal showed a negative correlation (r < −0.4) with sensory score. Trans, trans-2,4-nonadienal was detected in only two rice samples, more data were needed to verify the result that trans, trans-2,4-nonadienal was a characteristic volatile in rice.

39021608_p24

39021608

characteristic volatile compounds in rice

biomedical

Study

en

Combined the results of correlation analysis, OAV analysis and GC-O analysis, hexanal, 2-pentylfuran, octanal, 2-AP, 1-octen-3-ol, trans-2-octenal, decanal, trans-2-nonenal and trans, trans-2,4-decadienal were screened preliminarily as the potential characteristic volatiles. To further confirm the result, the absolutely OAVs of these compounds were obtained through their correlation factors which were determined according to Chinese Light Industry Standards . It was found that the OAVs of hexanal, 2-pentylfuruan, octanal, 2-AP, trans-2-octenal, 1-octen-3-ol, decanal, trans-2-nonenal and trans, trans-2,4-decadienal were greater than 1, and reached to 139.82, 2.35, 136.10, 10770.57, 11.56, 53.53, 9.42, 64.47 and 32.86, respectively. Hence, hexanal, 2-pentylfuran, octanal, 2-AP, 1-octen-3-ol, trans-2-octenal, decanal, trans-2-nonenal and trans, trans-2,4-decadienal were considered as the potential characteristic volatiles of rice odor, and analyzed in the following sensory analysis.

39021608_p25

39021608

characteristic volatile compounds in rice

biomedical

Study

en

The odor of volatiles often varied with contents . In order to elucidate the odor perception of characteristic volatiles and to explore the changes in volatile aroma caused by different contents, sensory ranking of odor intensity and preference level was performed at different contents. The range of volatile content in rice samples was obtained by multiplying the relative content range by a correction factor ( Table 2 ). Significant sensory differences in the odor intensity at different levels of content were found for all test volatiles except for 2-pentylfuran ( Table 2 ). For hexanal, octanal, 2-AP, and 1-octen-3-ol, there were significant differences in the odor intensity perceived within the content ranges of rice samples. The odor intensity of hexanal had significant differences among the content ranges of 23.56–50 ng/g, 50–100 ng/g, 100–500 ng/g and 500–699.09 ng/g . And significant differences in odor intensity between 0.5 and 5 ng/g and 10–79.78 ng/g, 1–100 ng/g and 50–570.84 ng/g, 0.5–10 ng/g and 50–80.53 ng/g were obtained for octanal, 2-AP, and 1-octen-3-ol, respectively. However, no significant differences in odor intensity were observed for trans-2-octenal, decanal and trans-2-nonenal and trans, trans-2,4-decadienal within the content range of rice samples. Table 2 F test values, odor descriptions and content range of volatiles. Table 2 volatiles F1 test F2 test content a (ng/g) Odor description hexanal 25.14 29.71 23.56–699.09 grassy, aged rice flavor 2-pentylfuran 6.06 3.20 1.14-13.64 green octanal 17.71 6.06 0–79.78 citrus 2-AP 20.69 9.94 0–570.84 popcorn, cooked rice trans -2-octenal 16.23 −6.29 4.92-34.69 unpleasant smell 1-octen-3-ol 11.89 9.49 0–80.53 mushroom, sweet decanal 9.26 7.11 0–28.26 fruity trans -2-nonenal 12.57 9.49 0–58.02 fat, green trans , trans -2.4-decadienal 12.11 16.11 0–2.53 fatty Notes: F1 test and F2 test were the F values of sensory intensity and preference level, respectively. a the content range of volatile in rice samples. Fig. 4 Sensory rank sum of odor intensity for volatile compounds at different contents. (a) hexanal; (b) 2-pentylfuran; (c) octanal; (d) 2-AP; (e) trans-octenal; (f) 1-octen-3-ol; (g) decanal; (h) trans-2-nonenal; (i) trans, trans-2,4-decadienal. Fig. 4

39021608_p26

39021608

The effect of characteristic volatiles on the aroma of rice

biomedical

Study

en

Moreover, the result of sensory ranking suggested that significant differences in preference level were found among the test contents for hexanal, 2-AP, 1-octen-3-ol, trans-2-nonenal and trans, trans-2,4-decadienal ( Table 2 ). However, no significant difference in preference level was observed for trans-2-nonenal and trans, trans-2,4-decadienal within the content range of the rice sample. The preference level decreased with the content of hexanal and 1-octen-3-ol increasing , indicating that hexanal and 1-octen-3-ol had a negative contribution to rice aroma. Furthermore, the negative contribution was also evidenced by the odor descriptions of hexanal and 1-octen-3-ol during sensory analysis, which were described as unpleasant grassy and aged rice flavor, and mushroom flavor. The preference level of 2-AP increased with the content, implying that 2-AP positively influenced the aroma of rice. Fig. 5 Sensory rank sum of preference level for volatile compounds at different contents. (a) hexanal; (b) 2-pentylfuran; (c) octanal; (d) 2-AP; (e) trans -octenal; (f) 1-octen-3-ol; (g) decanal; (h) trans -2-nonenal; (i) trans , trans -2,4-decadienal. Fig. 5

39021608_p27

39021608

The effect of characteristic volatiles on the aroma of rice

biomedical

Study

en

The perception of volatiles in rice matrix might be different from that without matrix, as the rice matrix was quite complex. To verify the influence of the characteristic volatile on rice aroma, each characteristic volatile was added to cooked rice samples, and consequently, triangle test was carried out. Triangle test was usually used to analyze whether there were perceptible differences between two samples. The result showed that the addition of hexanal, 2AP and trans, trans-2,4-decadienal caused significant changes in rice aroma (the number of correct selections were 24, 18 and 17, respectively). Moreover, according to the sensory description, adding 2-AP made the rice aroma stronger, while adding hexanal and trans, trans-2,4-decadienal made rice odor unpleasant. Therefore, an increase in 2-AP content in rice would improve the aroma quality of rice, and an increase in the contents of hexanal and trans, trans-2,4-decadienal would worsen the aroma quality of rice. Meanwhile, there were no significant perceptible differences between samples with and without adding 2-pentylfuran, octanal, trans-2-octenal, 1-octen-3-ol, decanal and trans-2-nonenal (the number of correct selections were 7, 10, 9, 10,11 and 9, respectively). Thus, to some extent, increasing in the contents of 2-pentylfuran, octanal, trans-2-octenal, 1-octen-3-ol, decanal and trans-2-nonenal would not cause significant perceptible changes in rice aroma.

39021608_p28

39021608

The effect of characteristic volatiles on the aroma of rice

biomedical

Study

en

Sensory ranking analysis showed that there were significant differences in the odor intensity of hexanal, 2-AP, octanal, and 1-octen-3-ol in the content range of rice samples. Moreover, significant differences in the preference level were observed for hexanal, 2-AP and 1-octen-3-ol, indicating significant influence of these volatiles on rice aroma. The result of triangle test showed that significant perceptible change in the aroma of cooked rice was observed after adding hexanal or 2-AP, further proving the significant effect of hexanal and 2-AP on rice aroma. Rice aroma increased by adding 2-AP and deteriorated by adding hexanal, indicating that 2-AP contributed positively to rice aroma while hexanal contributed negatively to rice aroma. Meanwhile, trans, trans-2,4-decadienal also had a negative effect on rice aroma as rice aroma was found to deteriorate after adding trans, trans-2,4-decadienal. Therefore, hexanal, trans, trans-2,4-decadienal and 2-AP were important characteristic volatiles for rice aroma. Their contents were supposed to have a great influence on the aroma quality of rice.

39021608_p29

39021608

The effect of characteristic volatiles on the aroma of rice

biomedical

Study

en

In this study, 85 volatile compounds were found in rice by gas chromatography-mass spectrometry analysis. Correlation analysis revealed that the volatiles negatively correlated with sensory score were positively correlated (r ≥ 0.35) with hexanal, indicating that hexanal could represent compounds negatively correlated with sensory score. GC-O analysis, OAV analysis and correlation analysis indicated that hexanal, 2-pentylfuran, octanal, 2-AP, 1-octen-3-ol, trans-2-octenal, decanal, trans-2-nonenal, and trans, trans-2,4-decadienal were potential characteristic volatiles for rice aroma. Meanwhile, the results of sensory analysis implied that hexanal, 2-AP, 1-octen-3-ol, trans-2-nonenal, and trans, trans-2,4-decadienal had significant effects on the aroma of rice. Among them, 2-AP was found to enhance the rice aroma, while hexanal, 1-octen-3-ol had a negative effect on the rice aroma. Moreover, it was found that addition of 2-AP significantly enhanced the aroma of rice while addition of hexanal and trans, trans-2,4-decadienal significantly deteriorated the aroma of rice. Their contents were supposed to have a great effect on the aroma quality of rice. Hence, hexanal, trans, trans-2,4-decadienal and 2-AP were proposed to be the key volatiles in future aroma evaluation. This study investigated the characteristic volatiles of rice and their effects on rice aroma, providing a reference for the evaluation of aromatic rice and amelioration of rice quality in the future.

39021608_p30

39021608

conclusion

biomedical

Study

en

Shuimei Li: Methodology, Investigation, Formal analysis, Validation, Data curation, Writing – original draft, Writing – review & editing. Hongyan Li: Investigation, Visualization, Validation. Lin Lu: Methodology, Validation, Resources. Gaoneng Shao: Resources, Methodology. Zhenling Guo: Investigation, Validation. Yuntao He: Investigation, Validation. Yong Wang: Resources, Supervision. Xiaohui Yang: Resources, Project administration. Mingxue Chen: Resources, Supervision, Project administration, Funding acquisition. Xianqiao Hu: Conceptualization, Methodology, Resources, Data curation, Project administration, Writing – review & editing.

39021608_p31

39021608

CRediT authorship contribution statement

other

Other

en

All the authors of this paper have approved the manuscript that is enclosed and no conflict of interest exists in the submission of this manuscript, and the contents of this manuscript have not copyrighted or published previously and is not under consideration for publication elsewhere.

39021608_p32

39021608

Declaration of competing interest

other

Other

en

Sixty patients with pterygium participated in the study. The pterygia patients were divided into primary and recurrent groups, and we collected conjunctival samples from 30 patients to use as a control group. Tan et al . graded pterygium according to tissue transparency and classified it into three types by using the visibility of episcleral vessels as a sign of translucency. Type 2 pterygiums were included in the current study based on the grading system of Tan et al .

38648458_p0

38648458

Methods

biomedical

Study

en