Biomimetic Mineralization of Gold Nanoclusters as Multifunctional Thin

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Biomimetic Mineralization of Gold Nanoclusters as Multifunctional Thin Films for Glass Nanopore Modification, Characterization and Sensing Sumei Cao, Shushu Ding, Yingzi Liu, Anwei Zhu, and Guoyue Shi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00802 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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Analytical Chemistry

Biomimetic Mineralization of Gold Nanoclusters as Multifunctional Thin Films for Glass Nanopore Modification, Characterization and Sensing Sumei Cao,† Shushu Ding,† Yingzi Liu,‡ Anwei Zhu,*,† Guoyue Shi*,† †

School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, People’s Republic of China ‡

Institute of Brain Functional Genomics, East China Normal University, 3663 Zhongshan Road N., Shanghai 200062, People’s Republic of China

E-mail: [email protected]

Tel: +86-21-54340042; Fax: +86-21-54340042

ACS Paragon Plus Environment


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Abstract Hurdles of nanopore modification and characterization restrain the development of glass capillary-based nanopore sensing platforms. In this article, a simple but effective biomimetic mineralization method was developed to decorate glass nanopore with a thin film of bovine serum albumin-protected Au nanocluster (BSA-Au NC). The BSA-Au NC film emitted a strong red fluorescence whereby nondestructive characterization of Au film decorated at the inner surface of glass nanopore can be facilely achieved by a fluorescence microscopy. Besides, the BSA molecules played dual roles in the fabrication of functionalized Au thin film in glass nanopore: they not only directed the synthesis of fluorescent Au thin film but also provided binding sites for recognition, thus achieving synthesis-modification integration. This occurred due to the ionized carboxyl groups (-COO−) of BSA coating layer on Au NCs which can interacted with arginine (Arg) via guanidinium groups. The added Arg selectively led to the change in the charge and ionic current of BSA-Au NC film-decorated glass nanopore. Such ionic current responses can be used for quantifying Arg with a detection limit down to 1 fM, which was more sensitive than that of previous sensing systems. Together, the designed method exhibited great promise in providing a facile and controllable solution for glass nanopore modification, characterization and sensing.


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Introduction Nanochannels or nanopipettes, called nanopores, offered an attractive platform for addressing a number of challenging questions in chemistry, biotechnology, and materials science.1-5 As one of newest and most exciting developments in this young field, the glass nanopipette-based nanopore platform is attracting more and more attention and has been applied for the study of metal ion detection,6,7 biomolecular interaction,8-11 protein detection,12,13 etc. Such nanopores have significant advantages of low cost, easily made, excellent mechanical and chemical stability, and adjustable orifice diameters. However, exploration of glass nanopores as sensors to identify the target or to study the interaction between the translocation target and the nanopore is still limited by the method to controllably modify their working region,14-16 which are believed to be very crucial for modulating the specificity and sensitivity of glass nanopores. Further stimulated by the desire for glass nanopores with rich surface functionalization chemistry, Au thin film decoration have emerged recently as one of the most promising tactics toward multifunction of the glass nanopores.17-19 Ultrathin gold-decorated conical glass nanopores were prepared for the first time through glucose oxidase-catalyzed reduction of HAuCl4.20 While this methodology to metallize glass nanopores can control the film thickness at a few nanometer scale, it required relatively lengthy and complicated functionalization process as well as expensive enzyme. Recently, a facile one-step photochemical method to prepare Au thin film-decorated glass nanopores have been developed with unsafe UV irradiation.21 Nevertheless, the obtained gold nanofilm inside single glass nanopore by



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both methods were only made up of Au particles of uniform composition rather than those of precisely controlled structure,22 and thus the uncontrollable interfacial chemistry of glass nanopore was unfavorable for the subsequent functionalization and may reduce the specificity and sensitivity of glass nanopores. In addition, to prove that the inner surface of the resultant glass nanopore was successfully modified with a thin gold film, a method favorable for the characterization of the morphology and the chemical compositions of the glass nanopore is essentially demanded. The characterization of gold modification to glass nanopore has been addressed by expensive and complicated techniques like transmission electron microscope (TEM), energy-dispersive X-ray (EDX) spectrum and elemental mappings.19-21,23 However, the glass nanopores, especially nanopores with orifice diameter less than tens of nanometers, were vulnerable to the electron beam irradiation of TEM. In a few seconds, the wall of glass tip melted and the Au thin film reformed into big Au particles or collapsed. Besides, to observe the gold nanofim as far as possible (the difference between gold film and glass is small in TEM image), the obtained high magnification TEM image only covered a small part of the tip, not being able to confirm the efficiency of functional modification in the entire nanopore tip zone. Moreover, the glass nanopores can no longer be used for sensing after TEM characterization because the tip of glass nanopore needs to be cut to meet the TEM sample preparation requirement, hence the research from nanopore structure to its property can hardly be performed. There is thereby an urgent need but it is still a significant challenge to develop a simple, low cost and especially


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nondestructive characterization method that offer improved resolution and greater field to visualize the Au film adsorbed onto the inner surface of single glass nanopore. Among the methods for achieving such a purpose, fluorescence observation and imaging by optical microscopy are particularly attractive.24-26 However, exploration of fluorescent approach to the direct characterization of gold film in glass nanopore have not yet been realized owing to the fluorescence quenching of dyes near the plasmonic Au nanostructures.27 Herein, we report a simple and green biomimetic mineralization method to decorate glass nanopore with structurally well-defined gold nanocluster films based on the capability of a common commercially available protein. The conical glass nanopore was first modified with poly(ethylene imine) (PEI) and bovine serum albumin (BSA) through a layer-by-layer electrostatic deposition route. Notably, there were three reasons for choosing BSA: (1) The attached BSA layer can sequester and encapsulate Au(III) ions followed by reduction of Au(III) ions through functional residues of BSA at pH 11.5,28 and finally a structurally well-defined BSA-Au nanocluster (BSA-Au NC) film was progressively formed in situ. (2) The BSA-protected Au NCs emitted an intense red fluorescence not only in solution but also in film. Besides, the BSA-Au NC film was stable in solution of a broad pH range and with a high salt concentration. The inherent advantagies of fluorescent BSA-Au NC film greatly facilitated their characterization by a simple, low cost and nondestructive fluorescence microscope. (3) Different from conventional two-step fabrication of glass nanopore sensors, namely, (i) Au film decoration at the inner surface of nanopore and (ii) gold surface modification



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for the introduction of recognition sites, by exploiting the ligands of BSA in this work, we first developed a “synthesis-modification integration” strategy for facile fabrication of functionalized Au thin film in glass nanopore. The ionized carboxyl groups (-COO−) of BSA coating layer on Au NCs can interact with arginine (Arg) via guanidinium groups, forming ion pairs through cooperative interactions.29,30 On the other hand, BSA has been used in the separation of enantiomers of amino acids.31 As a result, the glass nanopore decorated by BSA-Au NC film may directly be used for selective recognition of Arg enantiomers through monitoring of ionic current. To the best of our knowledge, it is the first report about BSA-directed synthesis of gold nanoclusters as multifunctional nanofilms for glass nanopore modification, characterization and sensing. In addition to proteins, this strategy can also be readily extended to other biologically relevant ligands ranging from single amino acids to linear peptides for the mineralization of noble metal nanoclusters and the modification of glass nanopores,32 and therefore will open great potential in the field of biosensing of glass nanopores.

EXPERIMENTAL SECTION Chemicals and Reagents. Linear polyethyleneimines (PEIs, Mw=25000) were purchased from Alfa Aesar. Gold(III) chloride trihydrate (HAuCl4·3H2O) and HEPES were supplied by Sinopharm Chemical Reagent Co., LTD. Bovine serum albumin (BSA), arginine (Arg), phenylalanine (Phe), leucine (Leu), valine (Val), alanine (Ala), threonine (Thr), glutamine (Glu), tyrosine (Tyr), aspartic acid (Asp), tryptophan (Trp),


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proline (Pro) were bought from Sigma-Aldrich. The aqueous solutions were freshly prepared using deionized water (18 MΩ·cm, Hitech science tool laboratory water purification system). Apparatus and Measurements. Scanning electron microscope (SEM) images were determined with a Hitachi S-4800 scanning electron microscope. To improve SEM image quality for the unmodified glass nanopore, an ultra-thin coating of platinum was applied onto the nanopore. Transmission electron microscope (TEM) images, energy-dispersive X-ray (EDX) spectrum and elemental mappings were taken by a JEOL JEM-2100 transmission electron microscope. TEM sample was prepared by placing a tip of glass nanopore on a folding grid. Imaging of the modified glass nanopore was performed on a Leica DMI3000 B inverted microscope (ex: BP 355-425 nm, em: LP 470 nm). Atomic force microscope (AFM) images were recorded by tapping mode on a Multimode 8 AFM from Bruker. Zeta potential of BSA-Au NCs was measured with a Malvern Zetasizer Nano ZS90. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry was acquired on an SCIEX TOF/TOF 5800 system. The oxidation state of BSA-Au NCs was monitored

by a

ThermoScientific

ESCALAB

250Xi

X-ray Photoelectron

Spectrometer (XPS). The fluorescence spectra were recorded on a Hitachi F-7000 fluorescence spectrophotometer. The absorption spectra were collected by a Shimadzu UV-1800 spectrophotometer. Fabrication of Glass Nanopores. Borosilicate capillary glasses (i.d. = 0.78 mm, o.d. = 1 mm) were bought from Sutter Instrument Co.. First, fresh piranha solution (3:1



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98% H2SO4/30% H2O2) was used to clean organic residues on glasses for about 2 h. It should be noted that piranha solution is an extremely powerful oxidizer with high corrosity. Hence, it must be prepared with great care. Then, the capillary glasses were washed with plenty of deionized water followed by vacuum drying at 80 oC. Finally, the glasses were pulled by a Sutter P-2000 micropipette puller with the following parameters: Heat = 320, Fil = 4, Vel = 40, Del = 180, Pull = 225. The inner diameter of the obtained nanopores could be assessed by TEM, SEM, and electrochemical measurements. Preparation of BSA-Au NC Film-Modified Glass Nanopores. First, 6 μL of 0.1 mg mL−1 PEIs solution was quickly backfilled into the tip of a freshly prepared conical glass nanopore using a micropipette syringe followed by tapping the nanopore to remove air bubbles. Then, the nanopore was left to stand for about 1.5 h to allow PEIs to interact thoroughly with the inner wall. After the removal of the excess PEIs solution, stabilization of the PEI coating inside nanopore was achieved by baking the glass nanopore at 50 oC for about 1 h. Next, 5 mL of 10 mM HAuCl4 solution was added dropwise to 5 mL of 30 mg mL−1 BSA solution under vigorous stirring. Two minutes later, 0.5 mL of 1 M NaOH solution was introduced dropwise and the obtained solution (pH ~11.5) was immediately filled in the PEIs-decorated nanopore. After that, the nanopore was placed at 70 oC for 2 h to allow the formation of thin film of BSA-Au NCs on the inner surface of nanopore. Finally, the excess solution was driven out to obtain the BSA-Au NC film-modified single glass nanopore. Ionic Currents Measurement. Ionic currents were measured by an HEKA EPC 10


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Double patch-clamp amplifier. Two Ag|AgCl electrodes were used to apply a voltage across the glass nanopore. One Ag|AgCl electrode inserting in the glass nanopore acted as a working electrode and the other Ag|AgCl electrode setting outside in the bulk solution worked as a reference/auxiliary electrode. Both the external bath solution and the solution backfilled in the nanopore were 0.03 M PBS (pH 7.0) unless otherwise indicated. The current−voltage (I−V) curves were obtained by scanning the voltage from −1 V to +1 V. Each test was repeated 5 times to obtain the average current value at a different voltage.

RESULTS AND DISCUSSION Fabrication of BSA-Au NC Film-Modified Glass Nanopores. The conical glass nanopores were first fabricated by a CO2 laser-based micropipette puller as described in the experimental section. From the current-voltage (I-V) response (Figure S1), the tip diameter of a typical glass nanopore was calculated to be ~65 nm using eq 1. It agreed well with the diameter estimated by the SEM (Figure S2). Because the inner surface of conical glass nanopore has charge, the obtained I-V curve showed nonlinearity. This phenomenon was called ionic current rectification (ICR),33 i.e., the nanopore shows an asymmetric current when applying a symmetric voltage. The degree of rectification in this work was quantified using the rectification coefficient r=ln|I+/I−|, in which I+ and I− were the value of current recorded at +1 V and −1 V. Since surface charge is one of the most important factors affecting ICR behavior,34,35 modification of new molecules inside glass nanopore can be monitored by acquiring r



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from the corresponding I-V curves. As expected, a negative current rectification (r =−0.71) was obtained on the unmodified glass nanopore because its surface was negatively charged in a neutral pH environment (Figure S3). When a monolayer of PEI with positive charge was adsorbed onto the inner surface of the nanopore through electrostatic interaction,36 the current rectification was switched to positive with r =0.25, as shown in Figure S3. This is because PEI bearing positively charged amino groups made the inner wall of nanopore positively charged in a neutral pH environment. Then, inspired by simple and green biomineralization processes,28 BSA was exploited for the preparation of Au film on the PEI-coated nanopore surface, because BSA can not only sequester and reduce Au precursors in situ to synthesize BSA-Au NCs at pH 11.5 but also adsorb onto the positively charged PEI monolayer at the same time (The Zeta potential of PEIs (0.1 mg/mL) at pH 11.5 was measured to be ~5 mV, Figure S4). Thus, an aqueous mixture of HAuCl4 and BSA with the solution pH adjusted to 11.5 was introduced to the PEI-decorated glass nanopore tip zone and incubated, as schematically shown in Figure 1. Note that the concentration of BSA, the reaction temperature and time had been optimized (Figure S5). The I-V curve of the obtained BSA-Au NC film-modified glass nanopores at pH 7.0 exhibited negative current rectification (r=−1.32, Figure S3), which was consistent with the fact that BSA was negatively charged in a neutral pH environment (pI≈4.6).37


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PEIs

PEIs

BSA

L-Arg

HAuCl4

D-Arg

BSA-Au NCs

L-Arginine

D-Arginine

Figure 1. Schematic diagram of the modification procedure of BSA-Au NC film-modified glass nanopore and its enantioselective recognition of ʟ- and ᴅ-Arg.

Characterization of BSA-Au NC Film-Modified Glass Nanopores. Then, to characterize the variation of the form and the chemical composition of conical glass nanopore after Au film decoration, TEM and energy-dispersive X-ray (EDX) spectrum were first tried. From the medium-magnification TEM image obtained in a very short time (Figure 2A), we can see that no obvious Au particles clumped or agglomerated at the inner surface of the modified nanopore. Instead, it was covered by a thin and smooth film, which had a different transparency as compared to the unmodified (Au-free) glass nanopore (Figure 2B). The EDX spectrum of the modified glass nanopore showed the peaks corresponding to Au elements (Figure 2C), indicating the presence of Au in the film. The uniformity of the gold deposition inside the nanopore tip was demonstrated by TEM elemental mappings (Figure 3). The outline of Au element distribution agreed with the glass nanopore shape as well as being a little narrower than that for Si and Al element from borosilicate glass. However, when obtaining high-magnification TEM images under strong electron beam irradiation, the wall of the glass nanopore tip melted and the nanopore size was enlarged rapidly (Figure S6). Meanwhile, the Au thin film in the nanopore was destroyed and reformed into big Au particles. Thus, the glass nanopores with larger



orifice diameter (~210 nm) were employed for TEM and TEM elemental mapping experiments in this work, although smaller sized Au-decorated glass nanopores can be fabricated. Note that the successful rate for the TEM characterization of such size (~210 nm) unmodified and Au-decorated glass nanopores was still very low even with great care.

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Cu Au

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Figure 2. (A,B) medium-magnification TEM images and (C,D) EDX spectrum of (A,C) the BSA-Au NC film-modified glass nanopore tip and (B,D) the unmodified (Au-free) glass nanopore tip. The inset is the high-magnification TEM image of Figure A.


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Figure 3. Dark-field TEM image (A) and the corresponding TEM elemental mappings (B-D: Si, Au, Al) of BSA-Au NC film-decorated glass nanopore.

Because it is challenging to characterize the inner surface of glass nanopore by TEM, to confirm that the designed synthetic route provided an efficient method for the metallization of glass nanopore, control experiments were performed in a flask containing the same reaction solution used for the Au modification of glass nanopores. A typical TEM image showed that the obtained BSA-Au NCs in solution were monodispersed with a size of around 1 nm (Figure S7A). The EDX spectrum also exhibited distinct peaks of Au element (Figure S7B). Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry showed intense peaks at m/z 66683 Da and 71578 Da for BSA and BSA-Au NCs (Figure S8), suggesting the existence of 25 gold atoms in the BSA-Au NCs. The in-depth chemical



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state of BSA-Au NC was studied by X-ray photoelectron spectroscopy (XPS). Two peaks at 83.8 and 84.4 eV assigned to the Au(0) and Au(I) were observed in the Au 4f7/2 spectrum (Figure S9A). The small amount of Au (I) (10.7%) helped to stabilize the Au NCs, as demonstrated in the structural study of other Au NCs.28 Due to their ultrafine size, the solutions of BSA-Au NCs emitted red fluorescence with emission peaks at 640 nm (Figure S5). Using fluorescein as a standard, the fluorescence quantum yield (QY) of BSA-Au NCs was calculated to be around 2.74%. These observations revealed that BSA can sequester and reduce Au ions in situ to prepare highly fluorescent BSA-Au NCs with red emissions. Notably, when a PEI-coated planar glass slide was exposed to the above reaction solution, Au thin film can be prepared on the PEI-coated glass surface simultaneously (Figure S10). This is because the dual role of BSA played in this work, it can not only biomineralized Au NCs but also adhered to the positively charged surface through its negative charges. In addition, BSA was beneficial for the stabilization of Au NCs by its cysteine residues and steric protection.28,38 In Figure S10, it can be clearly seen that the thin and smooth film adsorbed on the planar glass slide emitted strong red fluorescence, implying no obvious fluorescence quenching occurred in the assemblies of BSA-Au NCs as well as in its solid form. Furthermore, no significant changes of the fluorescence images and the absorption spectrum for the film were observed after treatment with high-salt solutions (up to 0.5 M PBS) (Figure S10, A-C, G) or solutions of a broad pH range (Figure S10, D-F, H), indicating that the BSA-Au NC film was very stable and can withstand high-salt buffer and pH variation.


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Apart from the high stability of BSA-Au NC film, the advantages of the glass nanopore modified with highly fluorescent Au film over other metalized glass nanopores are obvious: it can be facilely and quickly characterized by fluorescence imaging under a commercial fluorescence microscopy without requiring use of high demanding and expensive TEM-based techniques; and in contrast to a TEM image which covers a very small area on the specimen and does not have color, the fluorescence imaging can obtain a wider field of view to show the complete structure of conical glass nanopore and the picture was colored. To demonstrate the potential of fluorescence imaging in characterization of the internal structure of glass nanopore, a conical glass nanopore with a tip size of 70 nm was modified with BSA-Au NC film and then observed through a fluorescence microscopy. The fluorescence image (Figure 4B,C) reveals that the red fluorescent signals were localized at the inner surface of nanopore, implying that the fluorescent BSA-Au NCs were successfully adsorbed onto the inner wall of PEI-coated glass nanopore. Besides, it can be clearly seen in one image that a thin and smooth film featuring red fluorescence covered the entire inner surface of the nanopore tip zone, proving the designed protocol was competent to metalize single glass nanopore with long tip (>1 mm) and had a high level of effective modification. A control experiment showed no fluorescence for the PEI-coated glass nanopore (data not shown). The fluorescence scan in the conical glass nanopore further confirmed that the BSA-Au NC film maintained its emission characteristic (λex = 640 nm) in the inner wall of nanopore (Figure 4D). More importantly, the fluorescent BSA-Au NC film-modified glass nanopore can directly



be used after characterization whereas the TEM sample needs to cut the tip of glass nanopore and may easily be damaged by the electron beam irradiation,20 demonstrating the great potential of fluorescent conical glass nanopores in characterization and application. As far as we know, this is the first report to employ fluorescence imaging to characterize the morphology and the composition of conical glass nanopore.

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Figure 4. (A) The microscope image, (B) fluorescence microscope image, and (C) the overlay of microscope and fluorescence images of BSA-Au NC film-decorated glass nanopore tip (~70 nm) (D) Fluorescence emission scan (λex=405 nm) from three different regions of the BSA-Au NC film-decorated glass nanopore.


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Chiral recognition of Arg by glass nanopores. Modification of conical glass nanopore with BSA-Au NC film not only illuminated its internal surface but also provided a strategy to fabricate chiral recognition nanodevices. As is well known, BSA with enantioselectivity has been used in the separation of enantiomers of amino acids, small molecules and drugs.31,39,40 Compared with pure BSA molecules, the encapsulation of Au NCs in BSA molecules has little effect on the structure of BSA scaffolds, as noted in a structural study of BSA-Au NCs.28 Among amino acids, arginine (Arg) has the unique guanidinium residue which can form ion pairs with the ionized carboxyl groups of protein, 29,30 thus the BSA-Au NC film adsorbed on the glass nanopore may selectively bond more ʟ- or ᴅ-Arg enantiomer (pI 10.76) so the negative charge of Au film at neutral pH was reduced more for ʟ- or ᴅ-Arg enantiomer, therefore the change in ionic current is not the same. In glass nanopore, the negatively charged BSA-coated Au NCs adsorbed onto the positively charged PEI to form BSA-Au NC film and no obvious change occurred in the structure of BSA-Au NC in this process (the film maintained the same emission characteristic as the BSA-Au NC in solution). Hence, the BSA-Au NCs in solution as a surrogate for the Au film was tentatively used in zeta potential measurement to study the charge change of glass nanopore and the property of chiral Arg recognition in conical glass nanopore was assessed by ionic current. Initially, the zeta potential of BSA-Au NC (0.5 mg/mL, in 0.03 M PBS, pH 7.0) was about −11.9 mV (Figure 5A), so the BSA-Au NC film-modified glass nanopore presented a negative current rectification (Figure S3). When 0.12 mM ʟ- and ᴅ-Arg was added into 0.5 mg/mL BSA-Au NC solution, the



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zeta potential changed to about −2.44 and −6.50 mV. As compared to ᴅ-Arg, the charge of BSA-Au NCs decreased dramatically with increasing the concentrations of ʟ-Arg. This result demonstrated that the BSA molecules containing Au NCs may selectively interact with ʟ-Arg. Hence, the chiral Arg recognition ability of the BSA-Au NC film-modified glass nanopore was further studied through the ionic current changes of the BSA-Au NC film-modified glass nanopore. As shown in Figure 5B, when the modified glass nanopore was exposed to 1 pM ᴅ-Arg solution, the ionic current at −1 V decreased slightly, while if exposed to 1 pM ʟ-Arg, the ionic current at −1 V decreased remarkably. The current change ratios (defined here as the absolute value of the current change ratio at −1 V i.e., |(I-I0)/I0|) upon addition of 1 pM of ʟ- or ᴅ-Arg solution are 0.25 and 0.06, respectively (Figure 5C). In particular, the detection limit for ʟ-Arg could be achieved as low as 1 fM (Figure 5D), which was much better than other reported chiral detection methods, such as capillary electrophoresis and other electrochemical measurement.41,42 It is obvious that the BSA-Au NC film-modified glass nanopore exhibited chiral-selective ionic current response for Arg enantiomers.


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L-Arg-BSA-Au D-Arg-BSA-Au

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1.0

Concentration (pM)

Figure 5. (A) Zeta potential of 0.5 mg/mL BSA-Au NCs in the presence of different concentrations of ʟ- and ᴅ-Arg (0‒0.12 mM) and 0.03 M PBS (pH=7.0). (B) I-V characteristics and (C) ion current change ratios at −1 V of the BSA-Au NC film-modified glass nanopore (~68 nm) before and after addition of 1 pM ʟ- or ᴅ-Arg into 0.03 M PBS solution, pH=7.0. (D) Current-concentration properties of the BSA-Au NC-modified glass nanopore after the addition of different concentrations of ʟ- or ᴅ-Arg into 0.03 M PBS solution, pH=7.0. Each of the data point in (D) represents the averaging of currents at −1 V from five replicates of the BSA-Au NC

film-modified glass nanopores

with

addition of different

concentrations of ʟ- or ᴅ-Arg in PBS in sequence. Error bars in (A,C) represent standard deviations of measurements (n = 5).

To investigate how the inner wall of the resultant conical glass nanopore interacted with Arg enantiomers, there were several revealing observations. The potential contribution of PEIs on the chiral recognition of Arg was first studied. When the PEIs-coated glass nanopore was exposed to 1 pM ʟ- and ᴅ-Arg in PBS (pH=7.0)



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(Figure S11), there was very slight ionic current change, because both PEIs and Arg were positively charged, so adsorption of Arg by PEIs did not occur and thus there was no obvious change in the internal surface charge of the PEIs-coated nanopore. Then, the influence of Au core of BSA-Au NCs in chiral interaction was also investigated. The non-shifted absorption and fluorescence emission peak of BSA-Au NCs upon addition of Arg enantiomers gave strong evidence that there was neither interaction of the Au+ ions on the surface of Au NCs with Arg enantiomers nor etching of the Au NCs by Arg enantiomers (Figure S12).43-45 The XPS spectrum of Au 4f7/2 for the Arg-BSA-Au NCs also provided a supporting evidence (Figure S9B). When only BSA was adsorbed on the inner surface of PEI-decorated nanopore, if evaluated from the ionic current change degree (Figure S13), the nanopore exhibited the similar chiral recognition property as that for the BSA-Au NCs film-modified glass nanopore. As for the effect of pH on chiral Arg recognition, I-V curves of the BSA-Au NC film-modified glass nanopore before and after addition of ʟ- and ᴅ-Arg at different pH values were shown in Figure S14. At pH 10, no obvious current change was observed at −1 V in the presence of ʟ- or ᴅ-Arg because Arg has little positive charge at a pH value close to its isoelectric point (10.76), so the negative charge decrease of the modified film in the glass nanopore upon Arg binding was smaller. Besides, the chiral selectivity was reduced. At pH 7.3, the chiral Arg recognition property evaluated by ion current was consistent with the results shown in Figure 5B. At pH 2, although the protonated carboxylic groups (-COOH, pKa≈2) of BSA were neutral, BSA (pI≈4.6) was positively charged, so BSA may desorb from the PEI monolayer on the glass


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nanopore, and thus pH effect on chiral recognition was not investigated at pH 2. The effect of electrolyte concentration on the I-V behavior of the BSA-Au NC film-decorated glass nanopore was also investigated. With increasing the electrolyte concentration, the chiral selectivity of the glass conical nanopore decreased (Figure S15), because the shielding or competition by counterions against the association of between guanidinium and anionic groups increased and the diffuse double-layer thickness was reduced.30,33 The above results confirmed that Arg directly interacted with the BSA scaffold of Au NCs via the formation of ion pairs between the guanidinium group and ionized carboxyl groups (-COO−), thus the negative charge of the BSA-Au NC film was partially neutralized. So, the ionic current of BSA-Au NC film-decorated glass nanopore decreased by different degrees at neutral pH since much more ʟ-Arg was bonded with BSA compared with ᴅ-Arg. In addition to Arg, the selectivity of chiral recognition among other amino acids was also investigated. Compared with Arg, no obvious ionic current changes were observed for other amino acids even at 1 pM at neutral pH (Figure S16). Moreover, no chiral distinction between these amino acids enantiomers was achieved. That may because these amino acids do not have ionizable groups which are able to form ion pairs with the BSA-Au NCs film in the nanopore. Considering the isoelectric points (pI

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