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Multicolor Quantum Dot-Based Chemical Nose for Rapid and Array-Free Differentiation of Multiple Proteins Qinfeng Xu, Yihong Zhang, Bo Tang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03109 • Publication Date (Web): 13 Jan 2016 Downloaded from http://pubs.acs.org on January 21, 2016

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Analytical Chemistry

Multicolor Quantum Dot-Based Chemical Nose for Rapid and ArrayFree Differentiation of Multiple Proteins Qinfeng Xu,†, ‡ Yihong Zhang,†, ﹟

‡, §

Bo Tang*, ﹟ and Chun-yang Zhang*,﹟, ‡

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation

Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China ‡

Single-Molecule Detection and Imaging Laboratory, Key Lab of Health Informatics of Chinese

Academy of Sciences, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China §

Nano Science and Technology Institute, University of Science and Technology of China,

Suzhou, 215123, China * Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected]; [email protected].

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ABSTRACT Nanomaterial-based differential sensors (e.g. chemical nose) have shown great potential for identification of multiple proteins due to their modulatable recognition and transduction capability, but with the limitation of array separation, single-channel read-out, and long incubation time. Here, we develop a multicolor quantum dot (QD)-based multichannel sensing platform for rapid identification of multiple proteins in an array-free format within one minute. A protein-binding dye of bromophenol blue (BPB) is explored as an efficient reversible quencher of QDs, and the mixture of BPB with multicolor QDs may generate the quenched QD-BPB complexes. The addition of proteins will disrupt the QD-BPB complexes as a result of the competitive protein-BPB binding, inducing the separation of BPB from the QDs and the generation of distinct fluorescence patterns. The multi-color patterns may be collected at a singlewavelength excitation and differentiated by a linear discriminant analysis (LDA). This multichannel sensing platform allows for the discrimination of ten proteins and seven cell lines with the fastest response rate reported to date, holding great promise for rapid and highthroughput medical diagnostics.

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INTRODUCTION Rapid identification of disease-related proteins is imperative for biomedical research and clinic diagnostic.1-3 The antibody-based array, gel electrophoresis and mass spectrometry are frequently employed for specific protein profiling,4-6 but they involve either time-consuming procedures or expensive instruments. Alternatively, the chemical nose-based array enables rapid differentiation of multiple proteins in a cost-effective manner.7-8 Unlike antibody-/aptamer-based ‘lock-key’ specific recognition, the chemical nose-based array employs multiple differential receptors to generate protein-specific patterns for identification.9 The ideal receptors suitable for protein profiling should have maximal discrimination capability, involve minimal number of receptors, and generate response signal rapidly. Toward this goal, great efforts have been made to develop the receptor arrays by using a variety of scaffolds such as oligopeptides,10 porphyrins,11 micelle,12 polymer,13 DNA,14-15 and nanomaterials.16 Among them, nanoparticle-based receptors have got much attention due to their unique chemical/optical properties and tunable surface modifications.7-8 The differential nanoparticle receptors may be constructed by either one nanomaterial with diverse surface modifications17-18/complexations7-8,19-21 or multiple nanomaterials.22-23 The selective interaction between nanoparticle receptors and proteins may generate a protein-specific signature pattern which can be read out by either a direct17-18,22-23 or an indirect way through indicator displacement, 7-8,19-21 but the single-channel read-out can only measure the signals from spatially separated nanoparticle receptors. To simplify the array-based separation, some multifunctional nanomaterials are introduced for the production of multidimensional optical information.24-25 However, each optical signal still needs to be measured separately and the use of single nanoparticle receptor limits its discrimination capability. In addition, a long incubation time of ~ one hour is usually required to reach the equilibrium of nanoparticle-protein binding.78,19-21

To achieve the rapid identification of multiple proteins, the development of multichannel

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nanoparticle receptors is highly desirable. The multichannel nanoparticle receptors may transduce the signal in an array-free format without dependence upon nanoparticle-protein interaction.26-27 Quantum dots (QDs) have size-tunable narrow fluorescence emission and can be applied for simultaneous multiplexing analysis at a single excitation.28-29 The combination of multicolor QDs with different kinds of specific antibodies enables simultaneous detection of multiple proteins, but the involvement of different antibodies increases the experimental cost.30-31 Because the protein-dye complex absorbs the light at a wavelength different from the unbound dye, the protein-binding dyes can be used for the quantification of protein concentration, but the identification of a specific protein requires labor-intensive electrophoresis separation.32 Here, we develop a multicolor QD-based multichannel sensing platform for rapid identification of multiple proteins in a multichannel-sensing format within one minute. This sensing platform consists of three spectral resolvable QDs and a protein-binding dye of bromophenol blue (BPB), and the formation of triple-color QD-BPB differential receptors may result in the quenching of QD fluorescence by BPB. In the presence of target proteins, the high binding affinity of BPB with the proteins enables the competitive displacement of BPB quencher and consequently the recovery of QD fluorescence, generating distinct fluorescence patterns for protein identification. Ten proteins and seven cell lines may be discriminated by this multichannel sensing platform at a single excitation wavelength within one minute.

EXPERIMENTAL SECTION Materials and Measuremants. All quantum dot (QD) conjugates including streptavidin (SA) conjugates with (QD-SA-PEG: 525, 585, 655) / without PEG modification (QD-SA-ITK: 525, 605, 655), and the QDs modified with different organic groups modification (QD525-amino, QD585-carboxyl, QD655-biotin,) were obtained from Invitrogen (Carlsbad, CA, USA). The detailed characters of QDs including composition, quantum yields, full width at half maximum (FWHM), size, zeta potential and lifetime were shown in Table S1 (see Supporting Information). 4


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Immunoglobulin G (IgG), transferrin (Tf), fibrinogen (Fib), α1-antitrypsin (AAT), human lysozyme (Lyz), human α-thrombin (Tb), human serum, bromophenol blue (BPB), phenol red (PHR), bromocresol green (BCG) and iodophenol blue (IPB) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Human serum albumin (HSA) and α2-macroglobulin (A2M) were purchased from Prospec (Israel). Bovine serum albumin (BSA) was obtained from New England Biolabs (Ipswich, MA, USA). Human haptoglobin (Hp) was purchased from Abcam (Cambridge, MA, USA). All fluorescence measurements were performed on a spectrofluorometer (F-4600, Hitachi, Japan), and UV−Vis absorption was measured by a spectrophotometer (U-3900, Hitachi, Japan). The fluorescence lifetime was recorded on an Edinburgh FLS920 instrument with a 470-nm laser as the excitation source. The measurements of Zata potential and particle size were performed on a ZetasizerNano ZS (Malvern, Worcestershire, UK). Fluorescence titration of QDs by BPB. Each type of QDs at same concentration of 1.0 nM was separately titrated by BPB in 10 mM phosphate buffer (pH = 7.4). The initial fluorescence lifetime of QDs (200 µL, 1.0 nM) was measured in a micro quartz cuvette on a FLS920 spectrophotometer at the excitation wavelength of 470 nm. After the addition of aliquots of BPB in the cuvette, the fluorescence lifetime was measured again. The fluorescence lifetime value was calculated by exponential tail fit, normalized and plotted against various BPB concentrations. The binding constant (Kb) and binding stoichiometries (n) of QD-BPB complexes were obtained by nonlinear least-squares curve fitting analysis of the obtained plots using Origin 8.6 program (Origin Lab Co., Northampton, USA) based on the assumption that one QD possess n identical and independent binding sites. Differentiation assay of proteins in buffer. For the construction of triple-color differential receptors, three spectra-resolvable QDs and BPB were mixed in 10 mM phosphate buffer. Unless otherwise indicated, the triple-color QDs used in this research were modified by SA through a PEG linker (QD-SA-PEG), and the final concentrations of QD525-SA-PEG, QD585-SA-PEG, 5



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QD655-SA-PEG and BPB were 2.5 nM, 0.5 nM, 1.0 nM, and 3.5 µM, respectively. Taking into account the different fluorescence brightness of different-color QDs, the concentrations of various QDs were optimized to obtain same-level fluorescence intensities. Notably, BPB is a pH indicator and the QDs are susceptible to the changes of pH, ions, and microenvironment. In order to eliminate the interferences and ensure good reproducibility, it is necessary to use fresh detection solutions prepared by ultrapure water. With the addition of 49.5 µL of mixture solution into a cuvette, the fluorescence spectrum was scanned on an F-4600 spectrofluorometer in the range of 500 nm - 700 nm at the excitation wavelength of 400 nm. Then 0.5 µL of protein solution (100 µM) was added to the cuvette (final concentration of 1.0 µM) and mixed by pipetting. Without incubation, the fluorescence spectrum of QD-BPB complexes was scanned, and the intensity difference between these two measurements at the maximum emission wavelength was obtained. Six replicate experiments were performed for each protein sample. We further used two other groups of triple-color QDs (i.e., QD525-SA-ITK, QD605-SAITK, and QD655-SA-ITK; QD525-amino, QD585-carboxyl, and QD655-biotin) to investigate the effect of QD surface modifications upon the protein differentiation. The final concentrations of QD525, QD605 (or QD585), QD655 and BPB were 2.5 nM, 0.5 nM, 1.0 nM, and 3.5 µM, respectively. For the assay of unknown protein samples, the final concentrations of QD525-SAPEG, QD585-SA-PEG, QD655-SA-PEG and BPB were 2.5 nM, 0.5 nM, 1.0 nM, and 3.5 µM, respectively. The obtained data were subjected to a linear discriminant analysis (LDA) using SYSTAT 12.0 program (Systat, Richmond, CA, USA). Differentiation assay of proteins in human serum. For the differential sensing of proteins in human serum, the final concentrations of QD525-SA-PEG, QD585-SA-PEG, QD655-SA-PEG and BPB were 7.5 nM, 2.5 nM, 5.0 nM, and 14.0 µM, respectively. Previous research demonstrated that higher concentrations of fluorophore and quencher were required for the discrimination of multiple proteins in serum than in pure buffer.7, 20-21 Taking into account the 6


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high optical density of pure human serum, we used high-concentration QDs in this research to obtain high signal response. Because other proteins in serum may competitively bind with BPB, high-concentration BPB was used in this research as well. The obtained data were subjected to a linear discriminant analysis (LDA) using SYSTAT 12.0 program (Systat, Richmond, CA, USA). Cells differentiation assay. Seven cell lines were cultured in DMEM with 10% fetal bovine serum. For cell differentiation assay, the cells were washed with DPBS buffer for three times, and released with trypsin treatment. After the termination of trypsinization, the cells were spun down and resuspended in protein/antibiotics-free cell culture medium. Then the triple-color QD-BPB complexes were added into the cell suspension. The final concentrations of QD525-SA-PEG, QD585-SA-PEG, QD655-SA-PEG, BPB and cells were 2.5 nM, 0.5 nM, 1.0 nM, 3.5 µM, and 5000 cells / 50 µL, respectively. After incubation at 37°C for 1.0 min, the fluorescence spectra were measured. Six replicate experiments were performed for each cell sample. The obtained data were subjected to a linear discriminant analysis (LDA) using SYSTAT 12.0 program (Systat, Richmond, CA, USA). Calculation of binding constants by fluorescence titrations. According to previous report,33 the binding of BPB with QD may be described by equation 1 with an assumption that one QD possess n identical and independent binding sites. Kb Site + BPB ←→ Site ⋅ BPB

(1)

where Kb indicates the microscopic binding constant, and it can be calculated from equation 2.

Kb =

[ Site ⋅ BPB ] [ Site ][ BPB ]

(2)

where [Site·PBP], [Site] and [BPB] denote the equilibrium concentration of Site ⋅ BPB , Site, and BPB, respectively. Because the decrease of fluorescence lifetime (∆τ) results from the formation of Site ⋅ BPB , the concentration of Site ⋅ BPB may be calculated from equation 3 based on an assumption that Site ⋅ BPB is proportional to ∆τ.

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[ Site ⋅ BPB ] = ∆τ / m

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(3)

Where m denotes the proportionality coefficient which reflects the fluorescence difference of unit BPB before and after complexation. The [BPB] and [Site] may be calculated from equations 4 and 5 using their initial concentrations (i.e., [BPB]0 and [Site]0), respectively. [ BPB ] = [ BPB ]0 − [ Site ⋅ BPB ] = [ BPB ]0 − ∆τ / m

[Site] = [Site]0 − [Site ⋅ BPB] = n [QD]0 − [Site ⋅ BPB] = n [QD]0 − ∆τ / m

(4) (5)

The equation 6 may be obtained by the manipulation of equation 2 with equations 3-5. Kb =

∆τ / m ([ Site ]0 − ∆τ / m )([ BPB ]0 − ∆τ / m )

(6)

Solving this quadratic equation with one unknown (∆F) gives equation 7.

∆τ =

m ⋅ {([ BPB ]0 + n [QD ]0 + 1 / K b ) − ([ BPB ]0 + n [QD ]0 + 1 / K b ) 2 − 4n [ BPB ]0 [QD ]0 } 2

(7)

The binding constant (Kb) and binding stoichiometries (n) of various QD-BPB complexes may be obtained by nonlinear least-squares curve fitting analysis of titration data (i.e., ∆τ vs [BPB]) using equation 7 (Origin 8.6 program, Origin Lab Co., Northampton, USA).

RESULTS AND DISCUSSIONS

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Scheme 1. Principle of QD-based multichannel sensing platform for differentiation of multiple proteins. (a) Signal transduction strategy based on protein binding-induced displacement of bromophenol blue (BPB) quencher from the QD-BPB complex without the involvement of protein-nanomaterial interaction. (b) Workflow of multichannel sensing platform for differentiation of multiple proteins in an array-free format. The mixture of BPB with triple-color QDs induces the formation of three QD-BPB complexes with different stability. The addition of proteins may disassemble the QD-BPB complexes and restore the QD fluorescence. The distinct binding affinities between proteins and BPB may induce different BPB displacement patterns which are instantaneously reflected by triple-color fluorescence patterns for protein discrimination.

The principle of QD-based multichannel sensing platform is based on the competitive binding of small molecule BPB with the QDs and proteins (Scheme 1a). In the absence of proteins, the BPB binds non-covalently on the surface of QD, resulting in the quenching of QD fluorescence. Upon the addition of target proteins, the competitive protein-BPB binding disrupts the QD-BPB

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complex, inducing the separation of BPB quencher from the QD and consequently the recovery of QD fluorescence. The mixture of BPB with three spectrally resolvable QDs is exploited for the construction of differential nanoparticle receptors for multiple protein discrimination in a single solution (Scheme 1b). The binding affinity of BPB with different QDs is QD-dependent with the stability of QD-BPB complexes in the order of QD525 > QD585 > QD655 for QD-SA-PEG conjugates. With the addition of a protein with a weak binding affinity toward BPB (e.g., protein 1 in Scheme 1), only the BPB bound on QD655 surface may be displaced. In contrast, all BPBs bound on surface of triple-color QDs may be removed upon the addition of a protein with a strong binding affinity toward BPB (e.g., protein 3 in Scheme 1). Such protein binding affinitydependent BPB displacement may be reflected by corresponding triple-color fluorescence patterns, which may function as the unique fingerprints for protein differentiation. Owing to the unique optical properties of QDs,29 the number of receptors may increase up to 15, providing excellent discrimination capability for the identification of multiple protein.

Figure 1. The quenching of triple-color QD fluorescence by BPB (a-b) and its application to the detection of binding affinity (c) and HSA (d-f). (a) Normalized absorption spectrum of BPB and fluorescence emission spectra of triple-color QD-SA-PEG conjugates in the absence and in the 10


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presence of 2.2 µM BPB, respectively. (b-c) Fluorescence lifetime curves (b) and titration curves (c) of QD585 with the addition of BPB (■) and XF (●) at different concentrations. The blue scatter (▲) represents the fluorescence intensity titration and the red solid lines represent the nonlinear least-squares curve fitting analysis of titration data. (d-e) Fluorescence emission spectra (d) and time-resolved decay curves (e) of the mixture of QD525 (2.5 nM), QD585 (0.5 nM) and QD655 (1.0 nM) under various conditions: QDs (black line), QDs + BPB (blue line), QDs + HSA (purple line), QDs + XF (orange line), QDs + BPB + HSA (red line) and QDs + XF + HSA (olive line). The dark gray lines represent the fitting of lifetime data. The lifetime was measured at 585nm emission channel. The final BPB concentration is 3.5 µM, and the final HSA concentration is 1.0 µM. (f) Real-time monitoring of the kinetic behaviors of the binding of QD585 (0.5 nM) with BPB (1.4 µM) and the subsequent competitive displacement of BPB by HSA (2.0 µM).

We exploited the BPB as the protein-binding dye because of the rapid and reversible binding kinetics between proteins and BPB (the forward and backward rate constants are 4×108 mol.L-1.s-1 and 8×103 mol.L-1.s-1, respectively).34 BPB is a pH indicator and it has been widely used as a color marker to trace the gel electrophoresis process. The binding of BPB with proteins involves noncovalent dye-protein interactions such as hydrophobic interactions and electrostatic interactions.32 Especially, BPB has a strong absorption at 500-600 nm and may act as an efficient quencher of fluorescent dyes through fluorescence resonance energy transfer (FRET).35 In this research, we used three spectral resolvable QDs including QD525, QD585 and QD655, which are modified by streptavidin (SA) through a PEG linker (QD-SA-PEG), to investigate the quenching capability of BPB toward the QDs. The QD-SA-PEG conjugates have a protein-modified surface and may provide affinity sites for BPB. As shown in Figure 1a, upon the addition of 2.2 µM BPB, the fluorescence intensities of QD525 and QD585 decrease by as much as 96% and 90%, respectively. Despite of the poor spectral overlapping between QD655 emission and BPB

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absorption, the fluorescence intensity of QD655 decreases by as much as 82% (Figure 1a), suggesting that the quenching mechanism is not limited to only FRET. Because the highconcentration BPB shows a strong absorption toward 400 nm light (see Supporting Information, Figure S1), the fluorescence quenching may be partly attributed to the absorption of excitation light by BPB. To prove the quenching mechanism, we measured the fluorescence lifetime of QDs in the presence of different-concentration BPB. As shown in Figure 1b, the QD lifetime decreases with the increase of BPB concentration, excluding the possibility that the absorption of excitation light by BPB is a dominant factor of quenching. We further investigated the interaction of QDs with the unbinding dye xylene cyanol FF (XF) and various sulfophthalein dyes including bromophenol blue (BPB), phenol red (PHR), bromocresol green (BCG) and iodophenol blue (IPB) (see Supporting Information, Figures S2-S3). The maximum absorbance of BPB shows a blueshift with the removal of four bromine atoms (i.e., PHR) but a redshift with either the addition of two methyl groups (i.e, BCG) or the substitution of four iodine atoms for four bromine atoms (i.e., IPB), resulting in the variance of spectral overlapping between the QD emission and dye absorption and consequently the changes of quenching efficiency which can be calculated by (1-τ/τ0) ×100%. Based on the obtained quenching efficiency, we assumed that the quenching of QDs by BPB might be attributed to both energy transfer36 and heavy atom effect (bromine atoms), consistent with the previous research about the quenching of streptavidinmodified QDs by a protein binding dye of bromocresol green (BCG).37 -38 We further measured the binding constants of multicolor QD-SA-PEG-BPB complexes by nonlinear fitting of lifetime titration data (Figure 1c, see Supporting Information, Figure S3). Despite of the same surface modification, three different color/sized QDs exhibit different affinity toward BPB with the order of QD525 > QD585 > QD655 (see Supporting Information, Figure S3a). We also investigated two other groups of triple-color QDs: one group conjugated with same SA protein but without a PEG linker (QD-SA-ITK) and the other one modified with different organic groups (i.e., QD525-amino, QD 585-carboxyl, and QD655-biotin). Our results indicate 12


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that the QDs demonstrate color/size-dependent binding affinity toward BPB (Table 1, see Supporting Information, Figures S3a and S4) due to different hydrophobic groups and charge on the QD surface (see Supporting Information, Table S1). Specifically, the binding of SA-modified QDs with BPB involves the hydrophobic interaction and the electrostatic force; the binding of amino-modified QD525 with BPB involves the hydrophobic interaction and the electrostatic force due to the presence of positively charged amino group and the incomplete coverage of QDs by amphiphilic coating;39-40 whereas the binding of carboxyl modified-QD585 (or biotin modified-QD655) with BPB mainly involves the hydrophobic interaction. Therefore, we may construct multichannel sensing platform with differential receptors by simply mixing multicolor QDs with BPB.

Table 1. Binding constant (Kb) and Gibbs free energy (∆G) of different QD-BPB complexes. QD

Kb

-∆G

(×105 M-1) (kJ.mol-1) QD525-SA-PEG

8.33

33.8

QD585-SA-PEG

3.67

31.7

QD655-SA-PEG

1.80

30.2

QD525-SA-ITK

1.26

29.1

QD605-SA-ITK

0.88

28.2

QD655-SA-ITK

0.27

25.3

QD525-amino

8.62

33.8

QD585-carboxyl

0.12

23.3

QD655-biotin

1.51

29.5

Upon the addition of 1.0 µM human serum albumin (HSA), the fluorescence of free QDs remains unchanged, but the quenched fluorescence of triple-color QD-BPB complexes may be 13



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restored (Figure 1d). In contrast, HSA cannot induce the fluorescence recovery in the presence of unbinding dye XF, further confirming that the binding of HSA with BPB enables the separation of BPB from the QDs. Additionally, the maximum absorption of HSA-bound BPB exhibits a red shift of 13 nm as compared with that of free BPB (see Supporting Information, Figure S5), indicating the binding of HSA with BPB.41 Notably, when the absorbance value of QDs-BPB mixture at 400 nm decreases by 0.003, the corresponding transmittance of 400 nm excitation light increases by 0.62% (see Supporting Information, Figure S5). Because the observed fluorescence intensity is proportional to the intensity of excitation light, the increase of excitation light by 0.62% may only lead to the increase of fluorescence intensity by 0.62%, which is negligible for routine measurement of fluorescence intensity.

We further verified the principle of sensing platform using the time-resolved decay curves. The addition of HSA into the QDs-BPB mixture induces the increase of emission lifetime of QDs in 585 nm (Figure 1e), 525 nm, and 655 nm emission channels, respectively (see Supporting Information, Figure S6). Figure S7 shows a schematic illustration for the broken of triple-color QD-BPB complexes by competitive binding of HAS with BPB (see Supporting Information). We further investigated the kinetic behaviors of QD-BPB binding and the competitive displacement of BPB by HSA through real-time monitoring of QD585 fluorescence. At room temperature, the adsorption of BPB on QD surface and the HSA-induced desorption of BPB from QD surface are much rapid with the equilibrium being reached within 30 s (Figure 1f), suggesting that the multichannel sensing platform may provide an instantaneous signal response in a simply mixand-read manner.

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Figure 2. Discrimination of ten proteins by multichannel sensing platform. (a) Triple-color fluorescence patterns obtained by using triple-color QD-SA-PEG conjugates against ten proteins. (b-d) Canonical score plot of the triple-color fluorescence patterns obtained by using different QD-BPB receptors against ten proteins: (b) three QD-SA-PEG conjugates, (c) three QD-SA-ITK conjugates, and (d) the QDs with amino, carboxyl, and biotin modifications.

This multichannel sensing platform may be used to discriminate ten serum proteins including human serum albumin (HSA), bovine serum albumin (BSA), immunoglobulin G (IgG), transferrin (Tf), fibrinogen (Fib), α1-antitrypsin (AAT), α2-macroglobulin (A2M), haptoglobin (Hp), lysozyme (Lyz), and thrombin (Tb). These ten proteins have diverse structure/size, isoelectric point (pI), number of positively charged residues, and hydrophobicity/hydrophilicity (see Supporting Information, Table S2). As shown in Figure 2a, the addition of individual protein may induce the fluorescence recovery of quenched triple-color QD-BPB complexes, suggesting the stronger binding affinity of protein-BPB complex than that of QD-BPB complex. In each fluorescence channel, the recovered fluorescence intensity varies from the protein to the protein depending on both their hydrophobicity and the total number of positively charged residues.42-43

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Moreover, each protein generates a distinct triple-channel pattern (3 channels × 10 proteins × 6 replicates) which may function as a fingerprint for differentiation. The fluorescence response patterns (Figure 2a, see Supporting Information, Table S3) are subjected to a linear discriminant analysis (LDA), with the first two factors being plotted and visualized. Figure 2b demonstrates the clear clustering of ten distinct groups which correspond to ten proteins, with no overlap being observed (95% confidence ellipses), suggesting that this multichannel sensing platform can discriminate ten proteins. To investigate the effect of QD surface modification upon the protein discrimination efficiency, we investigated two other groups of triple-color QDs: one group conjugated with the same SA protein but without a PEG linker (QD-SA-ITK) and the other one modified with different organic groups (QD525-amino, QD585-carboxyl, and QD655-biotin). The obtained triple-color fluorescence patterns (see Supporting Information, Figure S8 and Table S4-S5) are converted to LDA score plots with 95% confidence ellipses (Figures 2c-d). The identification of ten proteins can be achieved by using both the QD-SA-ITK conjugate (Figure 2c) and the QDs modified with different organic groups (Figure 2d). Among above three groups, the SA-PEGmodified QDs possess the highest identification efficiency (Figure 2b), because the incorporation of PEG onto QD surface may significantly reduce the nonspecific protein adsorption. Notably, the stronger the binding affinity of QD-BPB receptors (Table 1), the higher the protein discrimination accuracy (see Supporting Information, Table S6).

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Figure 3. Identification of proteins by differential receptors which consist of triple-color QD-SAPEG conjugates and BPB under different conditions. (a) Canonical score plot of triple-color fluorescence patterns obtained by multichannel sensing platform against HSA at various concentrations (10 nM, 50 nM, 100 nM, 500 nM, 1 µM, and 5 µM). (b) Canonical score plot of triple-color fluorescence patterns obtained by multichannel sensing platform against the mixtures of HSA and IgG with total protein concentration of 2 µM. (c-d) Triple-color fluorescence patterns obtained by multichannel sensing platform against various proteins (5 µM) spiked in serum, (d) Canonical score plot of triple-color fluorescence patterns obtained by multichannel sensing platform against seven proteins.

We further used the multichannel sensing platform which consisted of triple-color QD-SAPEG-BPB complexes to identify 47 unknown protein samples. Out of 47 samples, 45 can be correctly classified (see Supporting Information, Table S7) with an identification accuracy of 95.7%, suggesting the good reproducibility of multichannel sensing platform and its feasibility in practical applications. In addition, the thermally denatured proteins can be easily distinguished

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from native proteins (see Supporting Information, Figure S9 and Table S8) due to the exposure of more hydrophobic residues induced by thermal denature. We further employed this multichannel sensing platform to detect HSA at various concentrations (Figure 3a, see Supporting Information, Table S9) and the mixtures of HSA and IgG (Figure 3b, see Supporting Information, Table S10). The HSA with as low as nanomolar concentration can be sensitively identified (Figure 3a), and a linear relationship between the first discriminant factor and the logarithm of HSA concentration is obtained (see Supporting Information, Figure S10). Moreover, pure HSA, pure IgG and four mixtures of HSA and IgG with different molar ratios can be distinctly distinguished in a LDA plot (Figure 3b), suggesting the excellent protein discrimination capability of multichannel sensing platform even in the mixtures. We further challenged this multichannel sensing platform with the discrimination of proteins in human serum. In order to get high fluorescence response in human serum, we used the higher concentrations of QDs and BPB in this research. Figure 3c (also see Supporting Information, Table S11) shows the obtained triple-color fluorescence patterns against seven proteins in human serum. Seven serum proteins can be identified correctly by LDA analysis with an accuracy of 100% (Figure 3d). Moreover, we demonstrated the identification of unknown proteins in serum sample with 94.3% identification accuracy (see Supporting Information, Table S12), suggesting the feasibility of multichannel sensing platform for real sample assay.

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Figure 4. Differential sensing of cells by multichannel sensing platform which consists of triplecolor QD-SA-PEG conjugates and BPB. (a) Signal transduction strategy based on the cell binding-induced displacement of BPB from the QD-BPB complex without the involvement of cell-nanomaterial interaction. (b) Triple-color fluorescence patterns obtained by multichannel sensing platform against seven cells (5,000 cells in 50 µL). (c) Canonical score plot of triple-color fluorescence patterns obtained by multichannel sensing platform against seven cells.

This multichannel sensing platform can be used for rapid identification of different cell types based on the differentiation of cell surface proteins.44 Because different cell lines have different surface proteins45 and the small molecule BPB cannot easily pass through the cell membranes,35 the instantaneous competitive displacement strategy may work for the differentiation of multiple cells. The presence of cells may disrupt the QD-BPB complexes, inducing the separation of BPB from the QD surface and consequently the recovery of QD fluorescence (Figure 4a). Three differential receptors consisted of triple-color QD-SA-PEG conjugates and BPB may generate distinct triple-color fluorescence patterns for the identification of specific cell type. As a proof of

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concept, seven human cells including one normal cell line (293T cells), and six cancerous cell lines (H460, H1299, A549, HepG, MCF-7, and Hela cells) (see Supporting Information, Table S13) are examined. Upon the addition of cells to the triple-color QD-SA-PEG-BPB complexes, different fluorescence recovery patterns are obtained within minutes (Figure 4b, see Supporting Information, Table S14). The LDA statistical analysis demonstrates that seven cells can be well discriminated with 95% confidence ellipses (Figure 4c). Moreover, an identification accuracy of 93.5% (29 out of 31) is achieved for the detection of 31 unknown cell samples (see Supporting Information, Table S15), suggesting the potentiality of multichannel sensing platform for clinic diagnosis.

CONCLUSION In summary, we have demonstrated that triple-color QD-BPB complexes may serve as highly effective differential receptors for instantaneous discrimination of multiple proteins. This multichannel sensing platform takes advantage of the unique optical properties of QDs as well as the rapid competitive displacement of small molecule dyes from QD surface by proteins, and it may generate the fastest response rate to date (see Supporting Information, Table S16). Ten serum proteins and seven cells can be identified correctly by using three spectra-resolvable QDSA-PEG conjugates without the involvement of spatial array separation. Moreover, the sensitivity and discrimination capability may be further improved by optimizing the protein-binding dye and increasing the number of spectral resolved QD receptors. This multichannel sensing platform enables rapid identification of multiple proteins and cells, holding great promise for highthroughput medical diagnostics.

ASSOCIATED CONTENT Supporting Information

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Supplementary Figures S1-S10 and Table S1-S16. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Tel.: +86 0531-86186033;

Fax: +86

0531-82615258.

E-mail: [email protected];

[email protected]. Author Contributions †

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grants 21325523, 21405169 and 21527811), the Award for One Hundred Talent Program of Nanyue of Guangdong Province, the Award for Pengcheng Distinguished Scholars of Shenzhen City, and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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