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In-situ Bio-conjugation and Ambient Surface Modification us-ing Reactive Charged Droplets Qing He, Abraham K. Badu-Tawiah, Caiqiao Xiong, Suming Chen, Huihui Liu, Yueming Zhou, Jian Hou, Ning Zhang, Yafeng Li, Xiaobo Xie, Jianing Wang, Lanqun Mao, and Zongxiu Nie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504111f • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Analytical Chemistry
In-situ Bio-conjugation and Ambient Surface Modification using Reactive Charged Droplets Qing He†, Abraham K. Badu-Tawiah*,‡, Suming Chen†,‡, Caiqiao Xiong†, Huihui Liu†, Yueming Zhou†, Jian Hou†, Ning Zhang†, Yafeng Li†, Xiaobo Xie†, Jianing Wang†, Lanqun Mao† and Zongxiu Nie*,† †
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA
ABSTRACT: Molecular ions are generated in induced electrospray ionization, and they can be transported to grounded ambient surfaces in the form of charged micro-droplets. Efficient amide bonds formation between amines and carboxylic acids were observed inside charged droplets during transfer to the surface. Biomolecules derivatized using this method were self-assembled on a bare gold surface via Au-S bonds under the charged micro-droplet environment. Cyclic voltammetric analysis of the self-assembled molecular film showed accelerated protein derivatization with cysteine, which allowed the covalent immobilization of the protein to the gold surface. Cytochrome C-functionalized electrodes prepared using the induced dual nano-electrospray process showed bio-activity towards aqueous solutions of hydrogen peroxide below 50 micro molar. In effect, we have developed a method that allows derivatization of biomolecules and their immobilization at ambient surfaces in a single experimental step.
Covalent immobilization of biomolecules on surfaces is of fundamental interest in bio-sensors, point-of-care applications, bio-interfacial science, nanotechnology, and materialogy.1-3 For example, the relatively simple instrumentation of redox protein bio-sensors (applicable in food, environmental, pharmaceutical, and clinical diagnostics) consist of a bio-recognition layer of protein attached to a transducer (the working electrode), and rely heavily on the appropriate choice of the immobilization method.4 Gold substrates are widely employed for biosensor applications because they provide both adsorption and covalent (including self-assembly monolayer (SAM) formation) immobilization means by which biomolecules can be anchored onto the (gold) working electrodes.5,6 A variety of commercial reagents are available for the preparation of functional gold electrodes (containing thiol, aldehyde, epoxy, etc. groups) that allow the formation of different types of covalent bonds using complementary functional groups such as thiol, amine, carboxylates, etc. present on the biomolecules.7-11 The formation of amide bonds is one of the most stable and widely executed techniques among bio-conjunction methods, and typically involves the reaction between activated carbonyl carbons and amines.12 The reaction time for this wet-chemistry based method is, however, long (at least 4 h) and involves three independent steps (scheme 1c): (1) attachment of a bi-functional linker containing thiol and carboxylic acid groups to the gold surface via
Au-S bond formation through surface self-assembly chemistry; (2) activation of the terminal carboxylic acid group through EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride) / NHS (NHydroxysuccinimide) chemistry; and (3) conjugation of the biomolecule through the reaction of the NHSactivated ester moiety (derived from step (2) above) with primary amine groups present in the biomolecule. The process can be achieved via a one-pot synthetic strategy, although it requires sequential addition of EDC, NHS and biomolecule in the presence of the modified surface generated from step (1). This study was motivated by the desire to reduce the reaction time for the bio-conjugation procedure with minimum activating reagents; this objective has advantages in green chemistry where the reduction/management of chemical waste is of major interest. We achieved these goals by performing the bioconjugation reaction under charged micro-droplet environment.13-15 Specifically, we have developed a new approach, based on induced electrospray,16,17 for molecule self-assembling on ambient, bare solid supports; the molecular film formation, and the in situ derivatization of biomolecules through the amide bond formation all occur in a single spray experiment. Charged droplets can be generated with (nano-) electrospray using direct current (DC) high voltage power
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supplies at either positive or negative charge state. However, in this experiment, we adapt the induced nanospray which employs alternation current (AC) high voltage to generating both positively- and negatively-charged droplets in the same plume. As will be shown, these droplets are more reactive than DC generated ones, and they enable in-situ one-step biomolecular derivatization and molecular self-assembly in less than 10 minutes. Herein, we use this AC spray plume in ambient soft landing experiment for the preparation of bio-active surfaces. The surface-contained bio-active molecules/film were characterized by cyclic voltammetry in the open laboratory environment (Scheme 1a). This methodology falls under the rubric of ambient (droplet) soft landing13-15, a recently developed mass spectrometry (MS) method that uses electrospray droplets to modify surfaces at atmospheric pressure, outsides the mass spectrometer.18 The vacuum counterpart of this soft landing (SL)19-24 experiment has previously been used to direct mass-selected biomolecular ions on to bare surfaces.25,26 In these previous vacuum-based SL experiments, the immobilization of bio-molecules usually occurred on previously prepared SAM surfaces. The SAM surfaces were routinely prepared by immersing the bare gold surface into a solution of dithiobis (succinimidyl undecanoate) to generate a semireactive surface, which allowed the immobilization of the bio-molecule via the formation of amide bonds (summarized in Scheme 1b).27,28 Gas-phase amide bond formation through ion/ion reactions29-31 insides the mass spectrometer has also been reported for peptide cross-linking and synthesis.27,28,32-34 Advantages of the atmospheric pressure droplet environment compared with the vacuum-based experiments include the facts that (1) ion generation, reaction and surface immobilization all occur in a single step, and (2) high ion currents can be utilized for experiments because of the absence of mass-selection during SL, although the latter advantage comes at a cost of our inability to selectively deposit a specific species from a complex sample. (3) There is also a growing interest to utilize the charged droplet environment for small-scale, combinatorial synthesis because of the accelerated reaction rates associated with the ambient charged droplet environment.13,35-39 Atmospheric pressure droplet reactions are attractive considering it is simpler and more effective to conduct chemical reactions compared with conventional wetchemistry based approach (scheme 1c). For example, rapid solvent evaporation from the smaller micro-droplets (radius ranges 1-5 µm)40-42 and/or thin liquid films created in an electrospray process has been found to cause moderate pH values in the starting solution to reach extreme values accompanied by increased reagent concentrations. These effects ultimately affords more effective collisions that results in enhanced rates for pH-catalyzed reactions.40 Moreover, the surfaces to be modified are completely accessible during experiments, and thus can be characterized using existing and traditional non-MS methods (e.g., atomic force and fluorescence microscopy, and surface enhanced Raman spectroscopy)14,18 without the need for
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instrument modification. In this study, we used cyclic voltammetric for surface characterization; this electrochemical detection method is simple, but it serves as a useful alternative for the expensive and commonly used methods, such as time-of-flight secondary ion mass spectrometry (TOF-SIMS) and infrared reflection absorption spectroscopy (IRRAS).27,28 Scheme 1. (a) Induced dual nano-electrospray experimental set-up. Biomolecules react with cysteine via EDC in the spraying droplets, and the resulting reaction products are anchored onto gold surface; this ambient spray methodology is compared with the multi-step (b) vacuum-based and (c) wet-chemistry based approaches
EXPERIMENTAL SECTION Prepared bare gold electrode were exposed to an induced dual-nanospray instrument for 10 minute at room temperature. Electrode was then rinsed with Milli-Q water to remove the physical adsorbed compounds. HHC|Cys|AuE was used for H2O2 detection with CV. Please see more details in Supporting Information.
RESULTS AND DISCUSSION Optimization of AC spray for surface modification The use of AC droplets (generated from the induced electrospray process)16,17 for immobilization of biomolecules on the gold surface was first investigated using cysteine as a model for compounds (ones that have an endogenous thiol group) that can be directly immobilized on gold without a prior derivatization step. In the induced electrospray experiment, there is no direct contact of the electrode with the solution of the analyte, and so providing long spray times without clogging the capillary spray tips. To immobilize cysteine (Cys) on gold surface, a solution of cysteine prepared in methanol/water (1:1, vol./vol.) was sprayed directly onto a grounded gold surface by applying an AC voltage (4 kV, peak to peak) to a stainless steel metal held in close proximity (0.5 mm) to the single-glass capillary containing the cysteine solution. The distance from the tip of the spray capillary to the grounded gold electrode was kept at 5 mm. As already explained, direct deposition of the elec-
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trospray plume at the gold surface allows rapid solvent evaporation, and thus concentrating cysteine molecules at the surface that facilitates Au-S bond formation. After 10 minutes of droplet deposition, the gold surface was washed with distilled water; the resultant cysteinefunctionalized gold surface (denoted as Cys|AuE) was characterized by X-ray photoelectron spectroscopy (XPS) and voltammetry. The XPS experiment showed the presence of thiol group anchored onto the gold surface (Figure S1, Supporting Information). The XPS data suggest a monolayer up to bilayer coverage of cysteine at gold surface (Figure S1, Supporting Information), something that might explain the redox activity observed in the cyclic voltammetric examination of the prepared Cys|AuE surface.43
Figure 1. Cyclic voltammogram of a) comparison of Cys|AuE prepared using bulk solution-phase reaction time of 100, 200, and 300 min (grey curves) versus 10 min of charged droplet deposition (black curve), b) Cys|AuE electrode obtained after 6 hours of bulk solution-phase reaction (dash), which is observed to be identical with the 10-minutes-spray Cys|AuE (solid), c) 10 cyclic voltammetric scans on charged-droplet modified Cys|AuE, after storing the electrode in air for 24 hours at room temperature.
In the cyclic voltammetric experiment (using phosphate buffered saline (PBS), 1X, pH 7.4), we found that the redox peaks (E1/2,ox = 0.185 V vs. SCE) generated from Cys|AuE electrode prepared from the 10 minutes spray time was consistent with redox peaks obtained from electrode prepared using 6 hours of bulk solution-phase reaction time (Figure 1a). Both anodic and cathodic peak currents of the spray-modified electrode varied linearly with scan rate (Figure S2, Supporting Information). Also, the ratio of the anodic peak current to the cathodic peak current was close to unity, indicating a surface-controlled electrochemical process.44 For solution-phase reactions, the gold electrode was placed in the cysteine solution and allowed to react for the desired reaction times of 100, 200 and 300 minutes (Figure 1b). The magnitude of cyclic voltammetric signal recorded from the resultant solutionphase derived Cys|AuE electrodes indicated that there is at least 30 times accelerated reaction rate for Au-S bond formation under the charged micro-droplet environment.
Stored in air for 24 hours at room temperature, the charged-droplet modified Cys|AuE electrode showed a considerable stability, even after 10 cyclic voltammetric scans (Figure 1c). Spray plumes derived from DC-based (nano-) electrospray or neutral spray (Figure S5, Supporting Information) were also efficient in the preparation of Cys|AuE as comparable redox peaks were observed in similar cyclic voltammetric experiments, suggesting that the high speed of droplets leads to an acceleration of the surface modification. Bio-conjugation in charged droplet environment Covalent immobilization of most bio-molecules on gold surface require prior bulk solution-phase derivatization of the bio-molecule with cysteine in the presence of EDC and NHS/Sulfo-NHS, at a suitable pH45,46. Under this solution-phase reaction conditions, Sulfo-NHS reacts with carboxylates in the presence of EDC to give a semi-stable Sulfo-NHS ester, which enhances the cysteinebiomolecule coupling process. This allows the indirect immobilization of biomolecules containing no thiol (-SH) functional groups on gold in a separate experiment. In this study, however, we performed biomolecular derivatization and immobilization in a single spray experiment. We first used glutamic acid to investigate the possibility of achieving derivatization (with cysteine) and immobilization in a single spray experiment. An induced dual nano-electrospray experimental set-up was employed (Scheme 1a). This set-up provides both positively- and negatively-charged droplets, within one continuous spray plume. In this methodology, 10 equivalent of EDC was mixed with 1 equivalent cysteine in one capillary, with no pH adjustment. The spray from this capillary results in the formation of O-acylisourea species. The second capillary was filled with 1 equivalent glutamic acid solution (see details in Supporting Information, section 2). We hypothesized that the rapid evaporation of oppositely charged droplets containing reactants can result in a violent infusion/mixing of the reactants into a limited small volume, which may improve reagent reactivity (see Supporting Information, section 2 for details). The resulting accelerated reaction products (a dipeptide consisting of cysteine and glutamic acid residues), which are contained in the spray droplets were directed onto a grounded gold surface for immobilization. In this case, we achieve indirect immobilization of glutamic acid by using cysteine as the linking/anchoring reagent. The resultant functionalized gold electrode is denoted as Glu|Cys|AuE. Cyclic voltammetric analysis of this Glu|Cys|AuE electrode using Cu(Ⅱ) buffer solution indicates that the reaction between glutamic acid and cysteine (in the absence of NHS) was successful in less than 10 minutes of spray time (Figure S7, Supporting Information). By contrast, the DC derived (nano-) electrospray and neutral spray droplets generated from a solution of glutamic acid, cysteine and EDC resulted in the formation of the standard Cys|AuE (Figure S8, Supporting Information). These results illustrate a relatively poor droplet reactivity when employing conventional DC-based / neu-
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tral spray, and that a thin film reaction at the surface has little effect in the derivatization reaction, suggesting that the bio-conjugation reaction had mostly occurred in the droplets before landing at the gold surface. We believe that the enhanced reactivity observed by infusing/mixing oppositely charged droplets derived from the induced dual nanospray apparatus is due to the neutralization of charge that results in the formation of a relatively longlived chemical complex in which the reactants can undergo intimate collisions.47 Ambient bio-active surface preparation We further applied the induced dual nano-electrospray device (Scheme 1a) for the immobilization of horse heart cytochrome C (HHC) to give a bio-active gold electrode (denoted as HHC|Cys|AuE), which was then used for the detection of hydrogen peroxide in aqueous solution. Native spray48-51 was used in this particular experiment in which cytochrome C was prepared in ammonium acetate buffer solution (pH 7.0) and sprayed using the induced dual nanospray apparatus. All attempts to apply DC nanoelectrospray and ESI did not result in the conjugation of cysteine to cytochrome C, and thus their inability to immobilize the HHC protein on gold electrode. Figure 2a shows a cyclic voltammogram (CV) obtained on HHC|Cys|AuE electrode prepared from a 10 minutes of spray time using the AC induced dual nanospray apparatus (Scheme 1a). Here too, a solution of cysteine and EDC was contained in one capillary, whereas the ammonium buffer solution of cytochrome C was contained in the second capillary. For purposes of comparison, CV spectra (Figure 2b) of electrodes prepared in bulk solution (with and without Sulfo-NHS added to solution) were also recorded. We designated HHC|Cys|AuE prepared by immersing Cys|AuE electrode in ammonium acetate buffer solution (pH 7.0) containing cytochrome C, EDC and NHS as our standard cytochrome C biosensor (Figure 2b, black trace); reaction time was four hours. The midpoint potential (E1/2,ox = 0.211 V and 0.214 V vs. SCE) and capacitance (Ah = 8.7 × 10-8 C and 9.4 × 10-8 C) obtained on the HHC|Cys|AuE electrode prepared by 10 minutes spray time was similar to that recorded for the standard HHC|Cys|AuE electrode (i.e., in bulk solution and with EDC and Sulfo-NHS included, Figure 2 b, black trace). By contrast, no redox peaks were observed for gold electrode (grey trace in Figure 2b) prepared using bulk solution containing only EDC and cytochrome C but with no added Sulfo-NHS, indicating limited derivatization of cytochrome C. We observed that a more intense and consistent redox peaks were obtained when the two spray capillaries were parallel and next to each other (tip-to-tip distance is about 2 mm), and by keeping the distance from spray tip to grounded gold surface at 5 mm. The AC voltage was applied at a distance of 0.5 mm from the side of the glass capillaries containing the solutions to be sprayed. The redox heme group in cytochrome C can show a peroxidase-like catalytic activity to reduce H2O2 in a giving sample52-54. Thus we employed the HHC|Cys|AuE elec-
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trodes prepared under the droplet environment to analyare 5 mL H2O2 samples in PBS at different concentrations using cyclic voltammetry. The electro-catalytic response varied linearly with increasing concentration of H2O2 (Figure 2c). These data suggest that minimal denaturation of cytochrome C occurred during the spray experiment, and that the electrode prepared from the charged droplet environment can be applied as an effective biosensor in detecting hydrogen peroxide.
Figure 2. Cyclic voltammogram of HHC|Cys|AuE prepared using a) 10 minutes induced dual nanoESI and b) 4 hours of bulk solution reaction with EDC and NHS (black line); Immersing in a solution with EDC only for 4 hours (grey line) gives practically no redox peaks. c) Calibration curve for detection of H2O2 from 0-50 μmol/L, error bar were taken from experiments at 3 individual times.
CONCLUSIONS In summary, molecular self-assembly at gold surface is readily achieved by directly delivering in situ derivatized biomolecular ions generated by reactive charged microdroplets onto a bare solid support under ambient conditions. The use of AC voltage in the induced dual nanoelectrospray experiment allowed the rapid generation and mixing of positively- and negatively-charged droplets that resulted in significant enhancement of the derivatization of cytochrome C/glutamic acid with cysteine, in the presence of EDC. No enhancement in the rate of the derivatization reaction was observed when using the conventional DC voltage- / neutral-based (nano-) electrospray setup. The charged micro-droplet environment should be applicable as a general method for in situ chemical derivatization and modification of ambient surface in the open laboratory, and under mild reaction conditions. The conversions of amino acids and proteins into the desired products were achieved in less than 10 minutes of spray time, and with limited used of (EDC) activating reagents. Bulk solution-phase reactions required the presence of both EDC and Sulfo-NHS in at least 4 hours of reaction time. Cytochrome C derived gold electrodes (HHC|Cys|AuE) were used as a biosensor for the detection of hydrogen peroxide showing the bio-activity of the protein at gold surface.
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ASSOCIATED CONTENT Supporting Information Experimental procedures, characterization data, mass spectra and other applications. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected]
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was supported by grants from the National Natural Sciences Foundation of China (Grant Nos. 21127901, 21321003, 21175139, 21305144 and 21205123), and Chinese Academy of Sciences. A.K.B.T. acknowledges funding from The Ohio State University start-up funds.
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In-situ Bio-conjugation and Ambient Surface Modification us-ing Reactive Charged Droplets Qing He, Abraham K. Badu-Tawiah, Caiqiao Xiong, Suming Chen, Huihui Liu, Yueming Zhou, Jian Hou, Ning Zhang, Yafeng Li, Xiaobo Xie, Jianing Wang, Lanqun Mao, and Zongxiu Nie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504111f • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Analytical Chemistry
In-situ Bio-conjugation and Ambient Surface Modification using Reactive Charged Droplets Qing He†, Abraham K. Badu-Tawiah*,‡, Suming Chen†,‡, Caiqiao Xiong†, Huihui Liu†, Yueming Zhou†, Jian Hou†, Ning Zhang†, Yafeng Li†, Xiaobo Xie†, Jianing Wang†, Lanqun Mao† and Zongxiu Nie*,† †
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA
ABSTRACT: Molecular ions are generated in induced electrospray ionization, and they can be transported to grounded ambient surfaces in the form of charged micro-droplets. Efficient amide bonds formation between amines and carboxylic acids were observed inside charged droplets during transfer to the surface. Biomolecules derivatized using this method were self-assembled on a bare gold surface via Au-S bonds under the charged micro-droplet environment. Cyclic voltammetric analysis of the self-assembled molecular film showed accelerated protein derivatization with cysteine, which allowed the covalent immobilization of the protein to the gold surface. Cytochrome C-functionalized electrodes prepared using the induced dual nano-electrospray process showed bio-activity towards aqueous solutions of hydrogen peroxide below 50 micro molar. In effect, we have developed a method that allows derivatization of biomolecules and their immobilization at ambient surfaces in a single experimental step.
Covalent immobilization of biomolecules on surfaces is of fundamental interest in bio-sensors, point-of-care applications, bio-interfacial science, nanotechnology, and materialogy.1-3 For example, the relatively simple instrumentation of redox protein bio-sensors (applicable in food, environmental, pharmaceutical, and clinical diagnostics) consist of a bio-recognition layer of protein attached to a transducer (the working electrode), and rely heavily on the appropriate choice of the immobilization method.4 Gold substrates are widely employed for biosensor applications because they provide both adsorption and covalent (including self-assembly monolayer (SAM) formation) immobilization means by which biomolecules can be anchored onto the (gold) working electrodes.5,6 A variety of commercial reagents are available for the preparation of functional gold electrodes (containing thiol, aldehyde, epoxy, etc. groups) that allow the formation of different types of covalent bonds using complementary functional groups such as thiol, amine, carboxylates, etc. present on the biomolecules.7-11 The formation of amide bonds is one of the most stable and widely executed techniques among bio-conjunction methods, and typically involves the reaction between activated carbonyl carbons and amines.12 The reaction time for this wet-chemistry based method is, however, long (at least 4 h) and involves three independent steps (scheme 1c): (1) attachment of a bi-functional linker containing thiol and carboxylic acid groups to the gold surface via
Au-S bond formation through surface self-assembly chemistry; (2) activation of the terminal carboxylic acid group through EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride) / NHS (NHydroxysuccinimide) chemistry; and (3) conjugation of the biomolecule through the reaction of the NHSactivated ester moiety (derived from step (2) above) with primary amine groups present in the biomolecule. The process can be achieved via a one-pot synthetic strategy, although it requires sequential addition of EDC, NHS and biomolecule in the presence of the modified surface generated from step (1). This study was motivated by the desire to reduce the reaction time for the bio-conjugation procedure with minimum activating reagents; this objective has advantages in green chemistry where the reduction/management of chemical waste is of major interest. We achieved these goals by performing the bioconjugation reaction under charged micro-droplet environment.13-15 Specifically, we have developed a new approach, based on induced electrospray,16,17 for molecule self-assembling on ambient, bare solid supports; the molecular film formation, and the in situ derivatization of biomolecules through the amide bond formation all occur in a single spray experiment. Charged droplets can be generated with (nano-) electrospray using direct current (DC) high voltage power
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supplies at either positive or negative charge state. However, in this experiment, we adapt the induced nanospray which employs alternation current (AC) high voltage to generating both positively- and negatively-charged droplets in the same plume. As will be shown, these droplets are more reactive than DC generated ones, and they enable in-situ one-step biomolecular derivatization and molecular self-assembly in less than 10 minutes. Herein, we use this AC spray plume in ambient soft landing experiment for the preparation of bio-active surfaces. The surface-contained bio-active molecules/film were characterized by cyclic voltammetry in the open laboratory environment (Scheme 1a). This methodology falls under the rubric of ambient (droplet) soft landing13-15, a recently developed mass spectrometry (MS) method that uses electrospray droplets to modify surfaces at atmospheric pressure, outsides the mass spectrometer.18 The vacuum counterpart of this soft landing (SL)19-24 experiment has previously been used to direct mass-selected biomolecular ions on to bare surfaces.25,26 In these previous vacuum-based SL experiments, the immobilization of bio-molecules usually occurred on previously prepared SAM surfaces. The SAM surfaces were routinely prepared by immersing the bare gold surface into a solution of dithiobis (succinimidyl undecanoate) to generate a semireactive surface, which allowed the immobilization of the bio-molecule via the formation of amide bonds (summarized in Scheme 1b).27,28 Gas-phase amide bond formation through ion/ion reactions29-31 insides the mass spectrometer has also been reported for peptide cross-linking and synthesis.27,28,32-34 Advantages of the atmospheric pressure droplet environment compared with the vacuum-based experiments include the facts that (1) ion generation, reaction and surface immobilization all occur in a single step, and (2) high ion currents can be utilized for experiments because of the absence of mass-selection during SL, although the latter advantage comes at a cost of our inability to selectively deposit a specific species from a complex sample. (3) There is also a growing interest to utilize the charged droplet environment for small-scale, combinatorial synthesis because of the accelerated reaction rates associated with the ambient charged droplet environment.13,35-39 Atmospheric pressure droplet reactions are attractive considering it is simpler and more effective to conduct chemical reactions compared with conventional wetchemistry based approach (scheme 1c). For example, rapid solvent evaporation from the smaller micro-droplets (radius ranges 1-5 µm)40-42 and/or thin liquid films created in an electrospray process has been found to cause moderate pH values in the starting solution to reach extreme values accompanied by increased reagent concentrations. These effects ultimately affords more effective collisions that results in enhanced rates for pH-catalyzed reactions.40 Moreover, the surfaces to be modified are completely accessible during experiments, and thus can be characterized using existing and traditional non-MS methods (e.g., atomic force and fluorescence microscopy, and surface enhanced Raman spectroscopy)14,18 without the need for
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instrument modification. In this study, we used cyclic voltammetric for surface characterization; this electrochemical detection method is simple, but it serves as a useful alternative for the expensive and commonly used methods, such as time-of-flight secondary ion mass spectrometry (TOF-SIMS) and infrared reflection absorption spectroscopy (IRRAS).27,28 Scheme 1. (a) Induced dual nano-electrospray experimental set-up. Biomolecules react with cysteine via EDC in the spraying droplets, and the resulting reaction products are anchored onto gold surface; this ambient spray methodology is compared with the multi-step (b) vacuum-based and (c) wet-chemistry based approaches
EXPERIMENTAL SECTION Prepared bare gold electrode were exposed to an induced dual-nanospray instrument for 10 minute at room temperature. Electrode was then rinsed with Milli-Q water to remove the physical adsorbed compounds. HHC|Cys|AuE was used for H2O2 detection with CV. Please see more details in Supporting Information.
RESULTS AND DISCUSSION Optimization of AC spray for surface modification The use of AC droplets (generated from the induced electrospray process)16,17 for immobilization of biomolecules on the gold surface was first investigated using cysteine as a model for compounds (ones that have an endogenous thiol group) that can be directly immobilized on gold without a prior derivatization step. In the induced electrospray experiment, there is no direct contact of the electrode with the solution of the analyte, and so providing long spray times without clogging the capillary spray tips. To immobilize cysteine (Cys) on gold surface, a solution of cysteine prepared in methanol/water (1:1, vol./vol.) was sprayed directly onto a grounded gold surface by applying an AC voltage (4 kV, peak to peak) to a stainless steel metal held in close proximity (0.5 mm) to the single-glass capillary containing the cysteine solution. The distance from the tip of the spray capillary to the grounded gold electrode was kept at 5 mm. As already explained, direct deposition of the elec-
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trospray plume at the gold surface allows rapid solvent evaporation, and thus concentrating cysteine molecules at the surface that facilitates Au-S bond formation. After 10 minutes of droplet deposition, the gold surface was washed with distilled water; the resultant cysteinefunctionalized gold surface (denoted as Cys|AuE) was characterized by X-ray photoelectron spectroscopy (XPS) and voltammetry. The XPS experiment showed the presence of thiol group anchored onto the gold surface (Figure S1, Supporting Information). The XPS data suggest a monolayer up to bilayer coverage of cysteine at gold surface (Figure S1, Supporting Information), something that might explain the redox activity observed in the cyclic voltammetric examination of the prepared Cys|AuE surface.43
Figure 1. Cyclic voltammogram of a) comparison of Cys|AuE prepared using bulk solution-phase reaction time of 100, 200, and 300 min (grey curves) versus 10 min of charged droplet deposition (black curve), b) Cys|AuE electrode obtained after 6 hours of bulk solution-phase reaction (dash), which is observed to be identical with the 10-minutes-spray Cys|AuE (solid), c) 10 cyclic voltammetric scans on charged-droplet modified Cys|AuE, after storing the electrode in air for 24 hours at room temperature.
In the cyclic voltammetric experiment (using phosphate buffered saline (PBS), 1X, pH 7.4), we found that the redox peaks (E1/2,ox = 0.185 V vs. SCE) generated from Cys|AuE electrode prepared from the 10 minutes spray time was consistent with redox peaks obtained from electrode prepared using 6 hours of bulk solution-phase reaction time (Figure 1a). Both anodic and cathodic peak currents of the spray-modified electrode varied linearly with scan rate (Figure S2, Supporting Information). Also, the ratio of the anodic peak current to the cathodic peak current was close to unity, indicating a surface-controlled electrochemical process.44 For solution-phase reactions, the gold electrode was placed in the cysteine solution and allowed to react for the desired reaction times of 100, 200 and 300 minutes (Figure 1b). The magnitude of cyclic voltammetric signal recorded from the resultant solutionphase derived Cys|AuE electrodes indicated that there is at least 30 times accelerated reaction rate for Au-S bond formation under the charged micro-droplet environment.
Stored in air for 24 hours at room temperature, the charged-droplet modified Cys|AuE electrode showed a considerable stability, even after 10 cyclic voltammetric scans (Figure 1c). Spray plumes derived from DC-based (nano-) electrospray or neutral spray (Figure S5, Supporting Information) were also efficient in the preparation of Cys|AuE as comparable redox peaks were observed in similar cyclic voltammetric experiments, suggesting that the high speed of droplets leads to an acceleration of the surface modification. Bio-conjugation in charged droplet environment Covalent immobilization of most bio-molecules on gold surface require prior bulk solution-phase derivatization of the bio-molecule with cysteine in the presence of EDC and NHS/Sulfo-NHS, at a suitable pH45,46. Under this solution-phase reaction conditions, Sulfo-NHS reacts with carboxylates in the presence of EDC to give a semi-stable Sulfo-NHS ester, which enhances the cysteinebiomolecule coupling process. This allows the indirect immobilization of biomolecules containing no thiol (-SH) functional groups on gold in a separate experiment. In this study, however, we performed biomolecular derivatization and immobilization in a single spray experiment. We first used glutamic acid to investigate the possibility of achieving derivatization (with cysteine) and immobilization in a single spray experiment. An induced dual nano-electrospray experimental set-up was employed (Scheme 1a). This set-up provides both positively- and negatively-charged droplets, within one continuous spray plume. In this methodology, 10 equivalent of EDC was mixed with 1 equivalent cysteine in one capillary, with no pH adjustment. The spray from this capillary results in the formation of O-acylisourea species. The second capillary was filled with 1 equivalent glutamic acid solution (see details in Supporting Information, section 2). We hypothesized that the rapid evaporation of oppositely charged droplets containing reactants can result in a violent infusion/mixing of the reactants into a limited small volume, which may improve reagent reactivity (see Supporting Information, section 2 for details). The resulting accelerated reaction products (a dipeptide consisting of cysteine and glutamic acid residues), which are contained in the spray droplets were directed onto a grounded gold surface for immobilization. In this case, we achieve indirect immobilization of glutamic acid by using cysteine as the linking/anchoring reagent. The resultant functionalized gold electrode is denoted as Glu|Cys|AuE. Cyclic voltammetric analysis of this Glu|Cys|AuE electrode using Cu(Ⅱ) buffer solution indicates that the reaction between glutamic acid and cysteine (in the absence of NHS) was successful in less than 10 minutes of spray time (Figure S7, Supporting Information). By contrast, the DC derived (nano-) electrospray and neutral spray droplets generated from a solution of glutamic acid, cysteine and EDC resulted in the formation of the standard Cys|AuE (Figure S8, Supporting Information). These results illustrate a relatively poor droplet reactivity when employing conventional DC-based / neu-
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tral spray, and that a thin film reaction at the surface has little effect in the derivatization reaction, suggesting that the bio-conjugation reaction had mostly occurred in the droplets before landing at the gold surface. We believe that the enhanced reactivity observed by infusing/mixing oppositely charged droplets derived from the induced dual nanospray apparatus is due to the neutralization of charge that results in the formation of a relatively longlived chemical complex in which the reactants can undergo intimate collisions.47 Ambient bio-active surface preparation We further applied the induced dual nano-electrospray device (Scheme 1a) for the immobilization of horse heart cytochrome C (HHC) to give a bio-active gold electrode (denoted as HHC|Cys|AuE), which was then used for the detection of hydrogen peroxide in aqueous solution. Native spray48-51 was used in this particular experiment in which cytochrome C was prepared in ammonium acetate buffer solution (pH 7.0) and sprayed using the induced dual nanospray apparatus. All attempts to apply DC nanoelectrospray and ESI did not result in the conjugation of cysteine to cytochrome C, and thus their inability to immobilize the HHC protein on gold electrode. Figure 2a shows a cyclic voltammogram (CV) obtained on HHC|Cys|AuE electrode prepared from a 10 minutes of spray time using the AC induced dual nanospray apparatus (Scheme 1a). Here too, a solution of cysteine and EDC was contained in one capillary, whereas the ammonium buffer solution of cytochrome C was contained in the second capillary. For purposes of comparison, CV spectra (Figure 2b) of electrodes prepared in bulk solution (with and without Sulfo-NHS added to solution) were also recorded. We designated HHC|Cys|AuE prepared by immersing Cys|AuE electrode in ammonium acetate buffer solution (pH 7.0) containing cytochrome C, EDC and NHS as our standard cytochrome C biosensor (Figure 2b, black trace); reaction time was four hours. The midpoint potential (E1/2,ox = 0.211 V and 0.214 V vs. SCE) and capacitance (Ah = 8.7 × 10-8 C and 9.4 × 10-8 C) obtained on the HHC|Cys|AuE electrode prepared by 10 minutes spray time was similar to that recorded for the standard HHC|Cys|AuE electrode (i.e., in bulk solution and with EDC and Sulfo-NHS included, Figure 2 b, black trace). By contrast, no redox peaks were observed for gold electrode (grey trace in Figure 2b) prepared using bulk solution containing only EDC and cytochrome C but with no added Sulfo-NHS, indicating limited derivatization of cytochrome C. We observed that a more intense and consistent redox peaks were obtained when the two spray capillaries were parallel and next to each other (tip-to-tip distance is about 2 mm), and by keeping the distance from spray tip to grounded gold surface at 5 mm. The AC voltage was applied at a distance of 0.5 mm from the side of the glass capillaries containing the solutions to be sprayed. The redox heme group in cytochrome C can show a peroxidase-like catalytic activity to reduce H2O2 in a giving sample52-54. Thus we employed the HHC|Cys|AuE elec-
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trodes prepared under the droplet environment to analyare 5 mL H2O2 samples in PBS at different concentrations using cyclic voltammetry. The electro-catalytic response varied linearly with increasing concentration of H2O2 (Figure 2c). These data suggest that minimal denaturation of cytochrome C occurred during the spray experiment, and that the electrode prepared from the charged droplet environment can be applied as an effective biosensor in detecting hydrogen peroxide.
Figure 2. Cyclic voltammogram of HHC|Cys|AuE prepared using a) 10 minutes induced dual nanoESI and b) 4 hours of bulk solution reaction with EDC and NHS (black line); Immersing in a solution with EDC only for 4 hours (grey line) gives practically no redox peaks. c) Calibration curve for detection of H2O2 from 0-50 μmol/L, error bar were taken from experiments at 3 individual times.
CONCLUSIONS In summary, molecular self-assembly at gold surface is readily achieved by directly delivering in situ derivatized biomolecular ions generated by reactive charged microdroplets onto a bare solid support under ambient conditions. The use of AC voltage in the induced dual nanoelectrospray experiment allowed the rapid generation and mixing of positively- and negatively-charged droplets that resulted in significant enhancement of the derivatization of cytochrome C/glutamic acid with cysteine, in the presence of EDC. No enhancement in the rate of the derivatization reaction was observed when using the conventional DC voltage- / neutral-based (nano-) electrospray setup. The charged micro-droplet environment should be applicable as a general method for in situ chemical derivatization and modification of ambient surface in the open laboratory, and under mild reaction conditions. The conversions of amino acids and proteins into the desired products were achieved in less than 10 minutes of spray time, and with limited used of (EDC) activating reagents. Bulk solution-phase reactions required the presence of both EDC and Sulfo-NHS in at least 4 hours of reaction time. Cytochrome C derived gold electrodes (HHC|Cys|AuE) were used as a biosensor for the detection of hydrogen peroxide showing the bio-activity of the protein at gold surface.
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ASSOCIATED CONTENT Supporting Information Experimental procedures, characterization data, mass spectra and other applications. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected]
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was supported by grants from the National Natural Sciences Foundation of China (Grant Nos. 21127901, 21321003, 21175139, 21305144 and 21205123), and Chinese Academy of Sciences. A.K.B.T. acknowledges funding from The Ohio State University start-up funds.
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