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Oct 1, 2015 - Institute of Molecular Medicine, College of Life and Health Sciences, Northeastern University, Shenyang, Liaoning 110819, China. §...
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Suspension Array of Ionic Liquid or Ionic Liquid−Quantum Dots Conjugates for the Discrimination of Proteins and Bacteria Shuai Chen,†,‡ Ling Wei,† Xu-Wei Chen,*,† and Jian-Hua Wang*,†,§ †

Research Center for Analytical Sciences, Northeastern University, Box 332, Shenyang, Liaoning 110819, China Institute of Molecular Medicine, College of Life and Health Sciences, Northeastern University, Shenyang, Liaoning 110819, China § Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, 300071, China ‡

S Supporting Information *

ABSTRACT: It is of great importance to develop novel and sensitive sensing materials for the detection of proteins and microorganisms to fulfill the demand of disease diagnosis. As the selectivity and sensitivity of sensing systems are highly dependent on the receptor, the fluorescent sensor array with imidazolium ionic liquids (ILs) and ionic liquid−quantum dots conjugates as semiselective receptors is developed for protein/bacteria differential sensing or discrimination. The IL sensing system formed by 1,3-dibutylimidazolium chloride (BBimCl), 1,3-diethylimidazolium bromine (EEimBr), 1,3-dibutylimidazolium bromine (BBimBr), 1,3-dihexylimidazolium bromine (HHimBr), and 1,3-dioctylimidazolium bromine (OOimBr) and the IL@QDs/QDs sensing system formed by CdTe, BBimCl@CdTe, EEimBr@CdTe, BBimBr@CdTe, and HHimBr@CdTe are tested, by transferring the interaction binding difference between receptors and proteins to the fluorescent response pattern. The IL sensing system is applied to the identification of 48 samples (8 proteins at 500 nM) with an accuracy of 91.7%. For the IL@QDs/QDs sensing system, 8 proteins are completely distinguished with 100% accuracy at a very low concentration level of 10 nM. Remarkably, 36 training cases (6 strains of bacteria from 3 different species) are discriminated with 100% (OD600 of 0.1).

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most attractive feature of the sensor array lies in selective rather than specific binding of sensing elements to the proteins. In practice, chemical sensors generally consist of two functional parts. The receptor is capable of converting the analyte information into a change in either physical or chemical property, while the transducer is a moiety that transforms and amplifies the perturbed properties into an observable analytical signal output, e.g., fluorescence, absorbance, and electrochemiluminesce.6 In most instances, the selectivity for a specific analyte originates from the receptor moiety. Recently, a number of receptor systems have been developed for array sensing purposes, e.g., charged dendritic phenylene-ethynylene fluorophores,8 DNA sequences,12 and aptamer systems.13 However, so far large-scale application of these sensing systems is still limited and their sensitivities are unsatisfactory for practical instances. In addition, one of the major limitations for the sensing systems is the lack of information regarding the analyte−receptor(s) interactions that facilitate the differentiation of analyte molecules.14 Ionic liquids have attracted extensive attentions and gained popularity in their interactions with proteins and triggered related investigations, e.g., the study of protein stability/activity, protein extraction, and separation/purification as well as protein crystallization.15,16 The fluorescence quenching of a

he detection of proteins, microorganisms/bacteria, biological cells, and other biosystems in complex sample matrices play a fundamental role in biomedical engineering and/or disease diagnosis. Polymerase chain reaction (PCR), enzyme-linked immunosorbent assays (ELISA), electrochemical assays, and other related techniques are the most widely used conventional approaches for clinical protein quantitation.1−4 Currently, various sensing technologies based on the detection of color or fluorescence signal change are widely employed in the field of biological sciences. However, the drawbacks of these technologies lie in the fact that simultaneous multianalyte assays (particularly in biological molecules) could not be achieved directly due to the limitation of binding interactions between the single lock and the corresponding key.5−10 In view of the growing demands on proteomics, medical diagnosis, and pathogenic detections, it is highly desired to develop novel sensing materials for the detection and discrimination of various proteins and microorganisms. Differential sensing has become an increasingly important concept in the field of supramolecular chemistry. Array-based methods have been demonstrated to be useful in addressing many sensing challenges. To mimic the mammalian olfactory system, differential sensing with a collection of semiselective sensors has been employed to provide a specific signal pattern for each single analyte or a group of analytes. The structurally similar analyte or a mixture of analytes is discriminated from others by chemometric analysis of a unique fingerprint.11 The © XXXX American Chemical Society

Received: July 1, 2015 Accepted: October 1, 2015

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Analytical Chemistry Table 1. Molecular Structures of the 5 Type Ionic Liquids Used as Sensing Receptors

Aesar Chemical Co., Ltd. (Tianjin, China). NaBH4 (96%), Cd(CH3COO)2·2H2O (99.5%), potassium tellurite (K2TeO3, 98%), and other chemicals are supplied by Sinopharm Chemical Reagent Co. (Shanghai, China). All the reagents are used as received without further purification. All the solutions are prepared with ultrapure water of 18.2 MΩ·cm. Preparation of CdTe Quantum Dots (QDs). CdTe QDs are prepared by following a previous method.21 Briefly, 0.0544 g of Cd(CH3COO)2·2H2O is dissolved in a round-bottom flask with 100 mL of deionized water. Eighteen μL of TGA is then added under stirring, and the solution is adjusted to pH 10.5 with NaOH solution (1.0 mol L−1). After stirring for 5 min, 0.0104 g of K2TeO3 dissolved in 100 mL of deionized water is introduced into the above solution, followed by the addition of 0.080 g of NaBH4 with further stirring for another 5 min. Afterward, the reaction mixture is refluxed at 100 °C under open-air conditions for 60 min. Finally, the CdTe QDs with desired photoluminescent emission property is obtained. Preparation of Ionic Liquid/Quantum Dots Conjugate (IL@CdTe QDs). Five types of ionic liquids as mentioned above, e.g., BBimCl, EEimBr, BBimBr, HHimBr, and OOimBr, are dissolved in 10 mL of CdTe QDs solution as previously treated giving rise to a final ionic liquid concentration of 0.1 mol L−1. The mixtures are then stirred at 400 rmp for 12 h under the dark at room temperature to produce the ionic liquid−quantum dots conjugates IL@CdTe QDs. Fluorimetric Assay of Proteins by the IL Sensing System. The protein sensing study is conducted by the following procedure: 200 μL of an ionic liquid sensing material

symmetrical imidazolium ionic liquid 1,3-butylimidazolium chloride (BBimCl) derived from the coordination between the cationic imidazolium moiety and iron atom in the heme group of hemoglobin offers a promising probe for the sensitive sensing of biomacromolecules.17 Ionic liquid itself as a part of chemical sensing material is insufficient for the selective discrimination, linear discriminant analysis (LDA), or principal component analysis (PCA) and has to be used to serve as an assistant for the discrimination between analytes. Ionic liquids can be readily adjusted by regulating their anionic or cationic moieties, and this high tunability is beneficial for the development of desired cross-receptors.18,19 In the present work, we attempt to develop a new sensing system for the discrimination of proteins, with ionic liquids or their conjugates with quantum dots as receptors based on the semiselectivity of ionic liquids toward protein species and the ionic liquid−protein binding/interaction behaviors.17,20

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EXPERIMENTAL SECTION Reagents and Materials. Hemoglobin (Hb), cytochrome c (Cyt-c), cysozyme (Lys), myoglobin (Mb), transferrin (Trf), ovalbumin (Ob), horseradish peroxidase (HRP), and bovine serum albumin (BSA) are obtained from Sigma-Aldrich (St. Louis, USA). 1,3-Dibutylimidazolium chloride (BBimCl), 1,3diethylimidazolium bromine (EEimBr), 1,3-dibutylimidazolium bromine (BBimBr), 1,3-dihexylimidazolium bromine (HHimBr), and 1,3-dioctylimidazolium bromine (OOimBr) are the products of Cheng Jie Chemicals Co., Ltd. (Shanghai, China). Thioglycolic acid (TGA, 90%) is purchased from Alfa B

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Analytical Chemistry solution (0.01 mol L−1 of BBimCl, EEimBr, BBimBr, HHimBr, or OOimBr) is loaded into a 0.5 mL tube. Then, 200 μL of blank solution (water) or a specific protein solution (500 nM of Hb, Cyt-c, Lys, Mb, Trf, Ob, HRP, or BSA) is added into the tube. After shaking at room temperature for 30 min, 200 μL of the mixed solution is transferred into a 96-well plate. The fluorescence intensity of the reaction mixture is recorded by a Biotek Synergy H1 Microplate Reader at 25 °C. For each sample solution, 6 replicate tests are performed with the measurement parameters (λex/λem and gain values) as listed in Table S1. Subsequently, the fluorescence differences between the readings with and without the addition of proteins are used as the fluorescence response. The raw data matrix is processed by using classical linear discriminant analysis (LDA) in SPSS (version 18.0). Fluorimetric Assay of Proteins by the IL@QDs/QDs Sensing System. The above 8 protein targets are tested with a IL@QDs/QDs sensing system formed by CdTe, BBimCl@ CdTe, EEimBr@CdTe, BBimBr@CdTe, and HHimBr@CdTe with the following procedure: 200 μL of one of the above sensing material solutions is loaded into a 0.5 mL tube, followed by the addition of 200 μL of either blank solution (water) or one of the 8 protein solutions (at various concentration levels of 500, 100, 50, and 10 nM). After shaking at room temperature for 30 min, the reaction mixture is transferred into a 96-well plate, and the fluorescence signal is recorded at λex/λem = 290/525 nm with a gain of 70 by the Microplate Reader at 25 °C. For each protein concentration, 6 replicate tests are performed with the measurement parameters (λex/λem and gain values) as listed in Table S1. The data matrix processing procedures are the same as those for the IL sensing system. For practical application, morning urine from a healthy volunteer is collected and centrifuged at 12 000 rpm to remove the insoluble matrix. The urine is 50-fold diluted before performing protein discrimination. Bacteria Identification. Bacteria are collected with inoculating loops from an agar slant culture medium, inoculated in broth, grown at 150 rpm and 37 °C for 10 h, and then stored at 4 °C for use. LB broth is used for Escherichia coli (BL21 and DH5α) and Bacillus subtilis (sortA and ars23), while a mixture of 10 mL of glucose (20%) and 90 mL of YPD is adopted for the yeast inoculation (W303 and EBY 100). Before the test, the bacteria are collected by centrifuging at 4000 rpm for 5 min, followed by washing with PBS buffer for three times. The bacteria solution is then adjusted to a final concentration of 0.1 and 0.01 OD600, respectively. Afterward, the solution is added to each sensing element (IL@QDs/QDs), and the variations of fluorescence intensity are used as the output response (λex/λem = 290/525 nm, gain = 70).

from the side chains. Eight proteins with different molecular weight (MW), isoelectric point (pI), and metal/nonmetal containing properties are used as the sensing targets (Table 2). Table 2. Basic Properties of the Eight Proteins Serving as Sensing Targets protein

MW (kDa)

pI

metal containing

hemoglobin (Hb) cytochrome c (Cyt-c) lysozyme (Lys) myoglobin (Mb) transferrin (Trf) ovalbumin (Ob) horseradish peroxidase (HRP) bovine serum albumin (BSA)

64.5 12.3 14.4 17.0 80.0 45.0 40.0 68.0

6.8 10.7 11.0 7.2 9.6 4.7 3.9 4.7

yes yes no yes yes no yes no

The constructed sensing system is first used for the identification of 8 proteins at the 500 nM level. In this case, the driving forces between the sensing element and the protein species include hydrophobic and electrostatic interactions associated with the variation of anionic moiety of the ionic liquid, as well as the interaction/coordination between the metal atom in protein and the imidazolium cation. The combination of these interactions results in the difference of entropy between the sensing elements (ILs) and the proteins. Consequently, the differential fluorescence responses would be obtained when various proteins interact with the 5 probes, i.e., the 5 ionic liquids. The differential relative fluorescence signal ratio from each single protein against the sensing system, i.e., (I − I0)/I0, forms a specific pattern for the discrimination of the protein from others by means of statistical analysis (Figure 1A).11 It shows that the fluorescence of ionic liquid is either quenched or enhanced after incubation with the 8 prtoeins. As previously demonstrated, an imidazolium ionic liquid 1,3butylimidazolium chloride (BBimCl) is highly fluorescent due to its unique symmetric plane conjugating molecular structure.17,20 The fluorescence of IL depends highly on the ambient condition. The fluorescence quenching of BBimCl in the presence of proteins is due to its tendency to remain in the hydrophobic cavity of proteins and thus exhibits a weak fluorescence in an aprotic environment,20 while fluorescence enhancement of HHimBr in the presence of proteins is due to its tendency to stay in the hydrophilic cavity of proteins and gives rise to a strong fluorescence in the protic environment.22 Similar trends are available for the other ionic liquids. The concentration dependent fluorescence quenching or enhancing behaviors of BBimCl and HHimBr by various proteins are given in Figure S1. By the employment of appropriate statistical analysis techniques, e.g., principal component analysis (PCA) and linear discriminate analysis (LDA), the resultant fluorescence response fingerprint can be quantitatively analyzed to achieve the protein identification and differentiation. Six replicates are tested for each protein sample, and the 240 raw data points (5 sensing materials × 8 proteins × 6 replicates) are subject to LDA analysis to generate five canonical factors (83.3, 9.6, 5.6, 1.4, and 0.2% of the variation), which represent linear combinations of the fluorescence response matrix. The first two most significant discrimination factors are employed to generate a 2D plot (Figure 1B) wherein each point represents the response pattern for an individual protein sample against the sensing system. Importantly, at a 500 nM concentration level of protein, we are able to cluster the 48 canonical

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RESULTS AND DISCUSSION Protein Discrimination with the IL Sensing System. We have previously demonstrated that ionic liquid 1,3butylimidazolium chloride (BBimCl) is highly fluorescent due to its unique symmetric molecular structure. In the present study, symmetric ionic liquid structures are used to construct a sensing system incorporating both receptor and transducer functions. For this purpose, 5 types of fluorescent ionic liquids with unique symmetric molecular configuration as given in Table 1, i.e., BBimCl, EEimBr, BBimBr, HHimBr, and OOimBr, are selected to construct the sensing system. These ionic liquids are chosen by following a criterion that they have different anions or different hydrophobic properties derived C

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Figure 1. (A) Fingerprints of 8 selected proteins based on the fluorescence response patterns of IL sensing system against the proteins at 500 nM. (B) Canonical score plot for the discrimination of the 8 proteins at 500 nM based on the IL sensing system.

fluorescence response patterns (8 proteins × 6 replicates) into 8 distinct groups with the classification accuracy of 91.7%. On the other hand, however, the canonical score plot demonstrates that only 4 out of the 8 protein targets are clearly identified in this pattern recognition and two groups of proteins (Ob and Mb, Cyt-c and Lys) still exhibit significant overlap and remain unidentified. These observations suggest that the IL sensing system is effective for the discrimination of protein mixtures under certain circumstances, while its discrimination capability is limited in some other cases. Protein Discrimination with the IL@QDs/QDs Sensing System. As described previously, weak fluorescence of some ionic liquids limited the performance of the 5-IL sensing system. For the purpose of improving the resolving capability of the sensing system, CdTe quantum dots are used to form conjugates with the ionic liquids employed in this study. The conjugates, shortened as IL@QDs, serve as new sensing elements and significantly improved the discrimination capability for the proteins by taking advantage of the high fluorescence of quantum dots and the IL@QDs conjugates. The experiments have shown that, although the fluorescence of IL@QDs conjugates is relatively lower than that for CdTe QDs itself (Figure 2), it is much increased with respect to those for the ionic liquids used in the IL sensing system, and the sensing

sensitivity or discrimination capability is significantly improved. It is noticeable that no florescence is recorded for the OOimBr@CdTe QDs conjugate, indicating that it is not suitable for serving as a sensing element. Considering that CdTe QDs are able to interact with proteins, i.e., HSA or Ob, and cause enhancement of the fluorescence due to the diminishing of surface defects,23,24 CdTe QDs are employed as one of the sensing elements to construct a 5-chanel IL@ QDs/QDs sensing array system with the other 4 IL@QDs conjugates, i.e., BBimCl@CdTe, EEimBr@CdTe, BBimBr@ CdTe, and HHimBr@CdTe. Figure 3A,B illustrates that the IL@QDs/QDs sensing array system is sufficiently sensitive for the identification of BSA at nanomolar level within a linear range of 0.01−5.0 μM. Linear responses of the other seven proteins are shown in Figure S2, i.e., 0.01−1.0 μM for Hb, 0.05−1.0 μM for Cyt-c, 0.01−1.0 μM for HRP, 0.01−1.0 μM for Lys, 0.01−1.0 μM for Mb, 0.01−1.0 μM for Ob, and 0.05− 1.0 μM for Trf. The detection limits for BSA, Hb, HRP, Lys, Mb, and Ob are derived to be 3.3 nM, while those for Cyt-c and Trf are 17 nM. The linearity of the dose−response curve suggests that the interactions between the IL@QDs/QDs sensing element and the protein species are homogeneous and stable. The usefulness of the IL@QDs/QDs sensing array system is evaluated by the identification/discrimination of the two groups of proteins, i.e., Ob/Mb and Cyt-c/Lys, which are unable to be identified by using the IL sensing system. It is surprising that these two groups of proteins are very well separated from each other by employing the IL@QDs/QDs sensing array system (Figure 4A). The results show that the classification/ discrimination accuracy is improved from 91.7% for the IL sensing system to 100% for the IL@QDs/QDs system. Meanwhile, all 8 proteins show significant separation in the 2D canonical score plot. The favorable performances in the sensing of proteins should be attributed to the interactions between the protein and the individual sensing element. The presence of various proteins results in the differential fluorescence responses due to their interactions with the QDs and IL@QDs probes (Figure 4B). The spectroscopic response patterns obtained from the fluorescence of each sensing element could categorize the 8 target proteins into three groups. When mixing and interacting with the 5 sensing elements, the first group of proteins including Hb, Cyt-c, and Mb containing a heme group cause a decrease of the fluorescence, while on the contrary, the second group of proteins, i.e., BSA and Trf, result in enhancement of the

Figure 2. Fluorescence emission spectra of CdTe and the related ionic liquid−quantum dots conjugates, including EEimBr@CdTe, BBimBr@CdTe, HHimBr@CdTe, and BBimCl@CdTe. D

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Figure 3. Identification of proteins at various concentration levels by using the IL@QDs/QDs system. (A) Canonical score plot for the fluorescence response patterns obtained with the IL@QDs/QDs sensing array system against BSA at 10 nM, 20 nM, 50 nM, 100 nM, 500 nM, 1 μM, and 5 μM for six replicate detections. Notably, since factor (2) is smaller than factor (1), it is possible by simply use factor (1) to identify the tested 8 proteins. In this study, the 2D plot is still employed for the consistency with other plots. (B) Plot of the discriminant factor (1) versus the logarithm of the BSA concentration.

Figure 4. (A) Canonical score plot for the discrimination of the 8 proteins at the 500 nM level based on the IL@QDs/QDs sensing system showing the discrimination very well. (B) Fingerprints of the 8 selected proteins at 500 nM based on the fluorescence response patterns of the IL@QDs/QDs sensing system. (C, D) Canonical score plot for the discrimination of the 8 proteins (C: 100 nM; D: 50 nM) based on the IL@QDs/QDs sensing system.

fluorescence. The remarkable difference of the first group of proteins from the other two groups lies in the presence of the heme group with the capacity of electron transport, and the fluorescence decrease/quenching phenomenon is a reasonable agreement with a previous conclusion.20 In addition, it is found that the IL@QDs/QDs sensing system is extremely sensitive to the protein conformation. LDA examination for the IL@QDs/ QDs response data at various protein concentrations, i.e., 50 and 100 nM, further reveals that all the 8 proteins exhibit significant separation in the 2D canonical score plot up to 50 nM (Figure 4C,D). Figure S3 demonstrates a canonical score

plot at the 10 nM concentration level of protein in the 2D canonical score plot; 6 out of the 8 protein targets are clearly identified in this pattern recognition, and only two proteins (Trf, Lys) still exhibit overlap. Nevertheless, the overlap could be ignored, as these proteins could be discriminated with identification accuracy of 100% at the 10, 50, 100, and 500 nM level (Tables S3−S6) with all 5 factors by LDA. Consequently, a limit of detection of 10 nM is achieved for the discrimination of the 8 protein targets by using the IL@QDs/QDs sensing system. For the sake of practical applications, it is more reliable E

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Figure 5. (A) Canonical score plot for the IL@QDs/QDs sensing array patterns as obtained from LDA for Hb and BSA at various concentrations (1000, 500, and 50 nM). (B) Protein identification in mixtures. Canonical score plot for the IL@QDs/QDs against protein mixtures (pure Hb; pure BSA; 10% BSA + 90% Hb; 30% BSA + 70% Hb; 50% BSA + 50% Hb). In each case, a total protein concentration of 1000 nM is employed.

case, each of the relevant proteins involved in the urine sample matrix generated a distinct response, and a 100% identification accuracy is achieved for all the 6 proteins. This observation indicated that the sensing array system based on QDs/IL-QDs conjugates offers promising potential for the discrimination of proteins in real biological samples. Bacteria Discrimination. Six strains of bacteria from three different species, i.e., Escherichia coli, yeast, and Bacillus subtilis, are chosen for demonstrating the discrimination capability of the IL@QDs/QDs sensing array system for processing complex samples. The basic properties of these bacteria, i.e., size and zeta potential, are listed in Table S7. By evaluating solely the differences of size and charge property, it is very hard to distinguish two strains of bacteria from each other. However, it is reasonable to speculate that the variations of protein species and their proportions on the surface of bacteria cells might provide a significant difference on the features for discrimination and give rise to different response patterns by adopting an appropriate sensing system. The IL@QDs/QDs sensing array system is used for the demonstration of this speculation. The 36 training cases are well separated into six respective groups with classification accuracy of 100% and 97.2% at bacteria concentrations of 0.1 OD600 and 0.01 OD600, respectively (Figure 7A,B). Reproducibility is a very important performance index for a sensing array system. In this respect, the different new growth bacterial strains have been tested 9 times to evaluate their discrimination. As listed in Table 3, there are 4 out of 9 with classification accuracy of 100% at 0.1 OD600, while there are 2 out of 9 with classification accuracy of 100% at 0.01 OD600 due to the lower concentration of samples. It is noteworthy that the discrimination accuracy for all the tests is >85%, indicating a favorable reproducibility of the IL@QDs/QDs sensing array system for bacteria identification. The observations described herein indicate that the present sensing array system exhibits potentials in the application for diagnosis of diseases, e.g., the identification of bacterial infections.

to perform the discrimination of the 8 protein targets at a higher concentration level of 50 nM. For a sensing array system, it is challenging to discriminate different proteins at various concentration levels and identify a mixture of proteins. The potentials of practical applications of the present sensing system, e.g., the identification of Hb and BSA at three concentrations, i.e., 50 nM, 500 nM, and 1 μM, are demonstrated. It is found that the LDA plots of various concentrations respond to certain patterns and could be well differentiated from each other (Figure 5A). Further experiments illustrated that the protein recognition capability of the IL@QDs/QDs sensing system is equally effective in the discrimination of protein mixtures, as demonstrated by the identification of 1 μM Hb and the BSA mixture with BSA/Hb molar ratios of 10/90, 30/70, and 50/50. Figure 5B indicated that these protein mixtures are clearly distinguished from each other in the LDA plot, by proper arrangement of the order of ratios in the dimension of the first factor. The performance of this multidimensional sensing system is further evaluated for the discrimination of proteins in human urine. For this purpose, the sensing array system is applied to control blank (water), urine, and urine samples spiked with relevant proteins, e.g., 500 nM of Hb, Cyt-c, Lys, Mb, Trf, or HSA. As shown in Figure 6, urine behaved differently with the control blank. It is known that the very complex matrixes in human urine is highly challenging for the identification of proteins and other biological targets.25 However, in the present

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CONCLUSIONS Ionic liquid as a new type of receptor has been demonstrated for the construction of a sensing array system for discriminating 8 proteins at 500 nM. In particular, the use of ionic liquid conjugates with quantum dots significantly improves the sensing sensitivity and discrimination accuracy. A detection limit of 10 nM is achieved for the discrimination of the 8

Figure 6. Canonical score plot for the discrimination of physiologically relevant proteins at the 500 nM level in the presence of human urine by using the IL@QDs/QDs sensing array system. F

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Figure 7. Identification/discrimination of bacteria with the IL@QDs/QDs sensing array system. Bacteria concentrations of (A) OD600 = 0.1 and (B) OD600 = 0.01 are used.

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Table 3. Reproducibility Results for Bacteria Identification

Corresponding Authors

occurring probability accuracy

OD600 = 0.1

OD600 = 0.01

36/36 35/36 34/36 33/36 32/36 31/36 30/36

4/9 0/9 1/9 3/9 1/9 0/9 0/9

2/9 1/9 0/9 3/9 0/9 2/9 1/9

*Tel: +86-24-83687659. Fax: +86-24-83676698. E-mail: [email protected]. *Tel: +86-24-83688944. Fax: +86-24-83676698. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors appreciate financial support from the Natural Science Foundation of China (21275027, 21235001, and 21475017), the SRFDP program (20120042110020), Liaoning Provincial Natural Science Foundation (2014020041), and Fundamental Research Funds for the Central Universities (N140505003, N141008001, N130105002).

protein targets by the IL@QDs/QDs sensing array system. The protein recognition capability of the IL@QDs/QDs sensing array system is reasonably effective for the discrimination of different proteins at various concentration levels and identification of protein mixtures. Six physiologically relevant proteins spiked in human urine are clearly identified, demonstrating that the QDs/IL-QDs conjugates offer a promising potential for the discrimination of proteins in real biological sample matrixes. Moreover, six strains of bacteria from three different species are well distinguished by the sensing array system with a 100% discrimination accuracy and favorable reproducibility. The IL@QDs/QDs sensing array system can serve as a readily accessible, highly discriminative, and adaptive tool for high-precision identification of biological targets. It also offers a new approach for the development of sensitive sensing arrays for specific biological or diagnostic purposes.

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AUTHOR INFORMATION

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02453. The operation parameters of the Microplate Reader; fluorescence response of proteins on BBimCl and HHimBr; linear response of 7 proteins by using the IL@QDs/QDs system; canonical score plot for the discrimination of the 8 proteins at the 10 nM level based on the IL@QDs/QDs system; detection results for the 8 proteins at 500 nM based on the IL sensing array system; detection results for the 8 proteins at 500, 100, 50, and 10 nM based on the IL@QDs/QDs sensing array system; basic properties of the target bacteria. (PDF) G

DOI: 10.1021/acs.analchem.5b02453 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.5b02453 Anal. Chem. XXXX, XXX, XXX−XXX