Double-Enzymes-Mediated Bioluminescent Sensor for Quantitative

Apr 19, 2017 - CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, CAS Key Laboratory of Standardization and Measurement for Na...
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Double-Enzymes-Mediated Bioluminescent Sensor for Quantitative and Ultrasensitive Point-of-Care Testing Yiping Chen,†,§ Yunlei Xianyu,†,§ Jing Wu,†,§ Mingling Dong,† Wenshu Zheng,† Jiashu Sun,*,†,‡ and Xingyu Jiang*,†,‡ †

CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, People’s Republic of China ‡ University of Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District, Beijing, 100049, People’s Republic of China S Supporting Information *

ABSTRACT: We report an ultrasensitive, quantitative, and rapid bioluminescent immunosensor (ABS) for point-of-care testing (POCT) of the disease biomarker in clinical samples using double enzymes including alkaline phosphatase (ALP) and luciferase. In the presence of the biomarker, the ALP attached on the surface of immuno-nanocomplex dephosphorylates adenine triphosphate (ATP), subsequently inhibiting the ATP−luciferin−luciferase bioluminescent reaction. The highly sensitive response of ATP (picomolar level) allows for ultrasensitive detection of biomarker via the effective change of the bioluminescence intensity through ALP- and luciferase-catalyzed reactions, which can be quantitatively determined by a portable ATP detector. This ABS fulfills the criteria for POCT that performs sensitive (femtomolar level of biomarkers) and quantitative measurement quickly (less than 1 h) with minimal equipment (portable detector).

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triphosphate (ATP), luciferase can catalyze the oxidation of luciferin into oxidized oxyluciferin and produce bioluminescence, and the bioluminescent intensity is proportional to the amount of ATP24−26 (Figure 1A). In this work, we use alkaline phosphatase (ALP, a widely used labeling enzyme in conventional immunoassays) to efficiently degrade ATP to adenosine monophosphate (AMP), which subsequently inhibits the luciferin−luciferase−ATP bioluminescent reaction and leads to the decrease of the bioluminescence intensity. The change of bioluminescence intensity (ΔBI) could be quantitatively determined by a portable ATP detector (Figure 1B). To construct a one-step and rapid immunoassay with the bioluminescent readout, we use antibody (Ab1)-conjugated magnetic nanoparticles (Ab1−MNPs) and polystyrene nanospheres conjugated with both ALP and secondary antibody (Ab2) (Ab2−PS−ALP). The presence of target could be captured both by the Ab1−MNPs and Ab2−PS−ALP to form the sandwiched PS−target−MNPs immuno-nanocomplex, which can be magnetically separated quickly. After the magnetic enrichment, ALP attached on the immuno-nanocomplex dephosphorylates the added ATP, which subsequently inhibits the ATP−luciferin−luciferase bioluminescent reaction. The luciferase-catalyzed reaction has a highly sensitive response of ATP (picomolar level) that allows for ultrasensitive detection of

ighly sensitive, quantitative, and straightforward point-ofcare testing (POCT) has greatly promoted the developments of clinical diagnosis,1−3 environmental monitoring,4 food safety inspection,5,6 and so forth.7 A successful POCT depends on three aspects: (1) simple operation and rapid reaction,8 (2) effective signal transduction9,10 and amplification,11−13 and (3) user-friendly and portable instruments for quantitative signal measurements. In recent years, portable instruments have been used as the readout system in the analytical strategies for detection of many types of targets.14,15 As one of the most widely used POCT devices, the glucose meter has been applied for assaying non-glucose targets with straightforward readout through the target-induced conversion of sucrose into glucose.16−18 The detection limit of non-glucose targets is at the nanomolar level due to the relatively low sensitivity of the glucose meter (at the millimolar level of glucose). To improve the sensitivity, additional steps for signal amplification need to be carried out.19−21 Although amplification can improve the sensitivity of analysis (improved by 1−2 orders of magnitude), their amplification effects are still restricted in many aspects. For example, the multistep signal amplification system based on click reaction increases the experimental complexity22,23 and prolongs the reaction time; thus, it is not suitable for POC format. To develop a highly sensitive POC assay with a simplified signal amplification step and straightforward readout, the sparkling fireflies at night give us the inspiration. The principle of firefly light emission is that, in the presence of adenosine © 2017 American Chemical Society

Received: January 19, 2017 Accepted: April 19, 2017 Published: April 19, 2017 5422

DOI: 10.1021/acs.analchem.7b00239 Anal. Chem. 2017, 89, 5422−5427

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108 U/L), and p-nitrophenyl phosphate disodium (pNPP, 10 mg) are from Sigma-Aldrich (U.S.A.). Anti-PCT capture monoclonal antibody (Ab1, 9.8 mg/mL) and anti-PCT detection monoclonal antibody (Ab2, 10 mg/mL) are from ABZYMO Biosciences (Beijing, China). Human IgG (10 mg/ mL), rabbit antihuman IgG (5 mg/mL), goat antirabbit IgG (10 mg/mL), and ALP-labeled secondary antibody are from Jackson (ImmunoResearch Laboratories, Inc.) We use phosphate-buffered saline (PBS) tablets from Amresco (U.S.A.) to prepare 0.01 mol/L phosphate-buffered saline (PBS, pH 7.4). We make PBST (0.01 M) by mixing PBS buffer (1000 mL) with 500 μL of Tween-20 (Amresco, U.S.A.). Human serum albumin and bovine serum albumin (BSA) are from Solarbio (Beijing, China). Other reagents and solvents are of analytical grade and from Beijing Chemical Reagents Co. (Beijing, China). We used deionizied water throughout all experiments. The portable ATP detector for bioluminescent signal readout is from Xi′an TianLong Science (Xi′an, China). A magnetic separation rack is from Shanghai Allrun Nano Science and Technology Co., Ltd. (Shanghai, China). We used an MS3 vortex oscillator (IKA Inc., Germany) to mix samples and other reagent solutions. Preparation of Ab1−MNPs. We transferred 500 μL of MNPs (10 mg/mL) into a tube and placed it on a magnetic separation rack for 1 min of separation. We carefully discarded the supernatant and resuspended the MNPs using 2 mL of double-distilled H2O. We next added 40 μL of EDC (20 mg mL−1) and 20 μL of sulfo-NHS (20 mg mL−1) into to the MNPs solution. After activation for about 30 min, we removed the excess EDC, NHS, and byproducts via magnetic separation using a magnetic separation rack. We then added 2 mL of PBS buffer (pH = 7.4, 0.01 M) to resuspend the activated MNPs. Subsequently, we added 0.1 mg of capture antibody (Ab1) into to the activated MNPs solution and gently stirred the mixture to react for 2 h at room temperature, followed by blocking the solution with 500 μL of 1% (m/v) BSA for 0.5 h. We obtained the Ab1−MNPs via magnetic separation step to remove the free Ab1 and used 1000 μL of PBS to resuspend the conjugate. The prepared solution was stored at 4 °C for further use. Preparation of Ab2−PS−ALP Conjugate. We transferred 400 μL of PS nanoparticle (5 mg/mL) into a 2 mL tube and centrifuged this nanoparticle at 8000 rpm for 5 min to remove supernatant. We then used 0.5 mL of deionized water to resuspend the PS nanoparticle. After resuspension, we immediately added 20 μL of the EDC solution (20 mg/mL) into the solution of PS nanoparticle. The mixture solution was gently stirred at room temperature for 15 min. We then added 1 mL of PBS solution (pH = 7.4, 0.01 M), 0.05 mg of detection antibody (Ab2), and 0.5 mg of ALP into the above solution, followed by incubation for 60 min at room temperature with gentle mixing. An amount of 100 μL of 5% BSA solution was added into the above solution, which was gently shaken at room temperature for 30 min. After this procedure, we centrifuged the mixture at 8000 rpm for 10 min and resuspended it in 1.5 mL of PBST solution, followed by centrifugation at 8000 rpm for 10 min. We repeated this washing step for three times. Finally, the Ab2−PS−ALP conjugate was resuspend in 1 mL of PBS (pH = 7.4, 0.1% BSA) and stored at 4 °C. Process of the ABS. We transferred 100 μL of MNPs-Ab1 (0.1 mg/mL), 100 μL of Ab2−PS−ALP (0.1 mg/mL), and 800 μL of different concentrations of targets into the individual 1.5

Figure 1. Scheme of the ABS for highly sensitive and quantitative detection of targets. (A) ALP can efficiently degrade ATP to AMP, which subsequently inhibits this bioluminescent reaction. In the presence of ATP, luciferase can catalyze the oxidation of luciferin into oxidized oxyluciferin and produce bioluminescence. (B) In ABS, the presence of target could be captured by the Ab1−MNPs and Ab2− PS−ALP to form the sandwiched PS−target−MNPs immunonanocomplex. After magnetic enrichment, the added ATP is dephosphorylated by ALP on the surface of PS, resulting in the change of the bioluminescence intensity (ΔBI) which is quantitatively measured by a portable ATP detector.

biomarkers via the effective change of the bioluminescence intensity. Compared with conventional bioluminescent immunoassay27 and other POCT strategies,28,29 this double-enzymesmediated bioluminescent immunosensor (ABS) has the following advantages: (1) It is highly sensitive due to the high sensitivity of this bioluminescent reaction responding to the change of ATP concentration (picomolar level); therefore, the target in samples can be effectively converted to the ΔBI signal through double enzymes (ALP and luciferase) catalyzed reactions. (2) The assay enables straightforward bioluminescent readout using the portable ATP detector (Figure S1), allowing for fast and convenient analysis. (3) Combined with Ab1− MNPs and Ab2−PS−ALP, the whole assay can be finished within 1 h, thus reducing the multiple washing steps in conventional immunoassays. (4)The assay has good stability because of the high enzymatic activity and stability of ALP as the labeling enzyme. These extraordinary features make the ABS promising for POCT.

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EXPERIMENTAL SECTION Materials and Equipment. Carboxyl-modified magnetic nanoparticles (MNPs) (200 nm in size; solid content, 10 mg/ mL) are from Ocean NanoTech (U.S.A.); carboxyl-modified polystyrene microsphere (PS) (300 nm, 5 mg/mL) are from Bangs laboratories, Inc. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), bioluminescent substrates adenosine 5′-triphosphate assay mix dilution buffer, adenosine 5′-triphosphate assay mix, adenosine 5′-triphosphate disodium salt hydrate, and alkaline phosphatase (ALP) (from bovine intestinal mucosa, 5423

DOI: 10.1021/acs.analchem.7b00239 Anal. Chem. 2017, 89, 5422−5427

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Analytical Chemistry mL centrifuge tubes separately. Each mixture was gently shaken for 30 min. We put all the tubes on a magnetic separation rack for 1 min and removed the supernatant. We then used 1 mL of PBST and 1 mL of distilled H2O to wash the mixture three times. After that, 200 μL of ATP solution (200 nM, Tris−HCl, pH = 8.5) was used to resuspend the MNPs−target−PS conjugate, and the mixture solution was gently shaken for 15 min at room temperature. After magnetic separation, the supernatant was mixed with 50 μL of luciferin−luciferase solution and we measured the bioluminescence signal quickly using the portable ATP detector. The limit of detection (LOD) is defined as follows: LOD = 3S/M; S, the value of the standard deviation of blank samples; M, the slope of standard curve within the low-concentration range. Selectivity of the ABS for Procalcitonin (PCT) Detection. We selected four other proteins [C-reactive protein (CRP), α fetal protein(AFP), carcino embryonie antigen (CEA), and human IgG] to evaluate the selectivity of the ABS for detection of PCT. The concentration of PCT is 0.1 ng/mL, and the concentration of other proteins is 1 ng/mL. Process of ATP-Mediated Enzyme-Linked Immunosorbent Assay (ELISA). We diluted the capture antibody using the carbonate buffer (0.2 M sodium carbonate/ bicarbonate, pH 9.6) and added 100 μL of diluted capture antibody into each well of a 96-well microplate. The plates were incubated at 4 °C for 12 h. After that, we washed the plates using PBST washing buffer (0.01 M PBS, with 0.5% Tween20). We then added 200 μL of blocking buffer (3% BSA) to each well for 2 h to ensure that all the remaining and available binding surfaces of the plastic wells were covered. After washing three times, we added the sample or target into each well, and the time of immunoreaction between antigen−antibody was 2 h. After washing four times, we added ALP-labeled secondary antibody into each well and put the whole plate at 37 °C for 1 h. Then, each well was washed three times with 1 mL of PBST and 1 mL of distilled H2O. We added 150 μL of ATP solution (50 nM, Tris−HCl, pH = 8.5) into each well, followed by incubation for 30 min at room temperature. Next, we added 50 μL of luciferin−luciferase solution into the above well and measured the bioluminescence signal quickly using the portable ATP detector. Real Sample Analysis. We collected serum samples (positive samples and negative samples) from Beijing Friendship Hospital after obtaining the patient-informed consent. Before analysis by the ABS, we diluted these samples 10-fold using Tris−HCl solution. Each sample was detected three times (n = 3).

Figure 2. TEM characterizations of immunomagnetic nanoparticles (MNPs, black) and polystyrene nanospheres (PS, gray) after magnetic separation, in the presence (A) or the absence (B) of rabbit antihuman IgG.

PS−ALP is crucial for the formation of immunocomplex which can be magnetically separated. The time of immunoreaction is within 15−30 min, because the immunoreaction between antibody and antigen is more rapid and sufficient in liquid phase than that at the solid−liquid surface. Response of the ABS to ATP and ALP. To investigate the efficiency of bioluminescent reaction, we first measure the response of ΔBI to the different concentrations of ATP. A linear relationship is observed between ΔBI and the concentration of ATP ranging from 0.5 to 1000 nM (Figure 3A). The lowest detectable concentration is 0.05 nM, and the linear range is over 3 orders of magnitude. For comparison, we

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RESULTS AND DISCUSSION Characterization of the PS−Target−MNPs ImmunoNanocomplex. To characterize the formation of sandwiched PS−target−MNPs immuno-nanocomplex, we use rabbit antihuman IgG as the target, which could bind to human IgG-conjugated MNPs (human IgG−MNPs) and PS conjugated both goat antirabbit IgG and ALP (goat antirabbit IgG−PS−ALP) by the specific antibody−antigen recognition. After magnetic enrichment, the transmission electron microscope (TEM) image reveals the coexistence of MNPs and PS linked through the target (Figure 2A). In the absence of target, we can only observe the MNPs by TEM after magnetic separation, indicating that no immuno-nanocomplex is formed (Figure 2B). These results suggest that the immunoreaction between human IgG−MNPs, target and goat antirabbit IgG−

Figure 3. (A) Linear response between ATP concentration and the ΔBI value. The concentration of ATP is from 0.5 to 1000 nM. (B) The sensitivity and linear range of the ABS and the pNPP-based optical method for detection of ALP in Tris−HCl solution (30% fetal calf serum). The concentration of ALP ranges from 0 to 8000 U/L. 5424

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IgG, a model protein, using the human IgG−MNPs and goat antirabbit IgG−PS−ALP. The ΔBI gradually increases when the concentration of rabbit antihuman IgG increases between 0.005 and 2000 ng/mL, and the LOD is 4.2 pg/mL (Figure 4C). The ΔBI has a linear response with the concentration of

study the analytical performance of the personal glucose meter.30,31 The sensitivity of the personal glucose meter for detection of blood glucose is at millimolar level, 6 orders of magnitude higher than that of the ATP meter for ATP detection (Table S1). The high sensitivity and broad linear range of the bioluminescent reaction ensure the improved performance of the ABS using the ATP detector for signal readout. We next investigate the correlation between ALP, ATP, and ΔBI, because ALP could degrade ATP and lead to the change of BI. The ΔBI increases with the increased concentration of ALP at the different concentrations of ATP (from 50 to 500 nM). The optimal concentration of ATP is 200 nM, which yields the maximum ΔBI (Figure S2A). We also study the matrix effects using four different buffer solutions (Tris−HCl, pH = 8.5; PBS, pH = 7.4; Na2CO3−NaHCO3, pH = 9.6; and pure water). With the increased concentration of ALP, ΔBI also increases in different buffer solutions. The ΔBI is maximum in Tris−HCl buffer (pH = 8.5), due to the high enzyme activity of ALP, and minimum in PBS solution (pH = 7.4), which inhibits the activity of ALP (Figure S2B). We should note that the enzyme activity of ALP is optimal at pH ∼ 9.5; however, the high pH value (Na2CO3−NaHCO3, pH = 9.6, 0.01 M) affects the activity and stability of luciferase, resulting in the lower ΔBI than that of Tris−HCl buffer (pH = 8.5). We thus choose 200 nM of ATP and Tris−HCl for the following ABS. Under the optimized conditions, we study the sensitivity and linear range of the ABS for detection of ALP. The ΔBI continuously increases with the increasing concentration of ALP from 3.75 × 10−3 to 8 × 103 U/L, and the limit of detection (LOD) is 3.5 × 10−3 U/L (LOD = 3S/M; S, the value of the standard deviation of blank samples; M, the slope of standard curve within the low-concentration range) (Figure 3B). The linear range is from 37.5 to 4 × 103 U/L of ATP (Figure 3B). For comparison, we also use the conventional pNPP-based optical method for ALP detection. The ΔOD405 continuously increases with the increasing concentration of ALP from 37.5 to 8 × 103 U/L, and the LOD is 32.5 U/L (LOD = 3S/M) (Figure 3B). A linear relationship between ΔOD405 and the concentration of ALP is from 3 × 102 to 4 × 103 U/L (Figure 3B). For ALP detection, the bioluminescent reaction exhibits higher sensitivity and wider linear range than pNPP, implying a good performance of the ABS which adapts ALP on the surface of PS to modulate the bioluminescent readout. In addition to the high sensitivity, the stability of the ABS is another important factor for POCT. We use the coefficient of variation (CV) to evaluate the stability of the ABS, and CV = (standard deviation/mean) × 100%. We investigate its stability from the following two aspects: (1) the stability of bioluminescent reaction at different concentrations of ATP, and (2) the stability of the ABS for response of different concentrations of ALP through the enzymatic reaction between ALP and ATP. The ATP−luciferin−luciferase bioluminescent reaction is stable, and the intra-CV and the inter-CV are both under 5% (Table S2), which ensure the stability of the bioluminescent signal. Moreover, the intra-CV and inter-CV at the different concentrations of ALP are both under 7% (Table S3). These results reveal the good stability of the ABS using the bioluminescent reaction and double-enzymes-mediated signal amplification. Detection of Model Protein. The ABS of high sensitivity and good stability is adapted for detection of rabbit antihuman

Figure 4. Sensitivity and linear range of the ABS, A-ELISA, and conventional ELISA for detection of rabbit antihuman IgG in Tris− HCl solution (30% fetal calf serum). (A) The scheme of conventional ELISA based on ALP for detection of rabbit antihuman IgG. (B) The scheme of A-ELISA for detection of rabbit antihuman IgG. (C) The standard curve and linear range of the ABS, A-ELISA, and conventional ELISA, respectively, for quantitative detection of rabbit antihuman IgG. The concentration of rabbit antihuman IgG is from 0 to 2000 ng/mL.

rabbit antihuman IgG in the range between 1 and 1000 ng/mL (Figure 4C). For comparison, we employ conventional ELISA (Figure 4A) and ATP-mediated ELISA (A-ELISA) (Figure 4B) for detection of this model protein. The A-ELISA uses the ALP-conjugated secondary antibody to replace the nanoparticles (MNPs and PS). The LODs of conventional ELISA and A-ELISA are 0.84 ng/mL and 45 pg/mL, respectively (Figure 4C). The linear range for IgG detection is 5 and 500 ng/mL by A-ELISA and 10−250 ng/mL by conventional ELISA (Figure 4C). Among the three methods, the ABS exhibits the best performance for rabbit antihuman IgG detection with improved sensitivity and linear range, attributed to its double enzyme (luciferase and ALP) mediated bioluminescent system and the use of human IgG−MNPs and goat antirabbit IgG−PS−ALP. Conventional ELISA only relies on the ALP-catalyzed enzymatic amplification as the readout, so 5425

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ELISA for PCT detection. In conventional ELISA, the ΔOD405 increases when the concentration of PCT is from 10 to 105 pg/ mL. The linear response of PCT detection is 10−104 pg/mL, and the LOD is 8.4 pg/mL by conventional ELISA (Figure 5, parts A and B). Thus, the sensitivity of the ABS for detection of PCT has been improved by 2 orders of magnitude compared with conventional ELISA. Meanwhile, the linear range of the ABS is 1 order of magnitude broader than that of conventional ELISA. The selectivity test of the ABS for PCT detection shows that ΔBI from the PCT measurement is greater than that from other proteins (Figure S3), suggesting that this strategy has good specificity due to the highly specific immunorecognition. In addition, we investigate the intra- and inter-CV of the ABS for detection of spiked PCT at different concentrations in PBS solution with 3% BSA (Table S4). The intra-CV at the different concentrations of PCT are under 8.4%, and inter-CV at the different concentrations of PCT are under 10.7%, which suggest that the reproducibility of this assay is good enough to satisfy the need for detection of picogram per milliliter level of biomarkers. We further employ the ABS for PCT detection in the real serum samples. The PCT levels in serum samples are predetermined by the clinical diagnostic method, the electrochemiluminescence immunoassay with an automated analyzer [Roche-ECL (E601), Roche Diagnostics]. The clinical cutoff value of PCT detection is 100 pg/mL. All of the five negative samples (samples 1−5) are detected to be negative by both the ABS and conventional ELISA. All of the positive samples (samples 6−20) are identified to be positive by the ABS method, but samples 10−12 and 18−20 are detected to be negative by conventional ELISA due to the insufficient sensitivity (Figure 5C and Table S5). These results indicate that the ABS has a higher accuracy than that of ELISA for PCT detection. Moreover, the measured PCT levels by the ABS show good consistency with the Roche-ECL (Figure 5D and Table S6). With the same accuracy, the ABS is much more portable than the Roche-ECL, showing great potential in POCT. We also compare the analytical performance of the ABS with related reported systems that involve only one enzyme or one kind of nanoparticle, including the reproducibility, detection limit, and linear range (Table S7). These related works include the surface plasmon resonance-based immunoassay33 and electrochemical immunosensor34 for detection of PCT and some related bioluminescent immunoassays35−37 for detection of other targets. The reproducibility of the ABS is as good as these methods (Table S7), and the sensitivity and portability of the ABS for detection of PCT are better than those of other methods. The ABS has three advantages. (1) High sensitivity: the LOD of the ABS for detection of disease biomarker can reach the femtomolar level, while that of other POCT methods is at the picomolar or nanomolar level. (2) Fast reaction: the ABS employs Ab1−MNPs and Ab2−PS−ALP to construct a homogeneous strategy, dramatically shortening the time of the one-step immunoreaction within 30 min. (3) Straightforward, quantitative, and rapid readout: the commercialized and handheld ATP detector has a rapid response (10 s) to the bioluminescent signal.

the sensitivity is inferior to that of the ABS and A-ELISA. AELISA uses commercialized ALP-labeled secondary antibody that only conjugates one or two ALP molecules. In comparison, one goat antirabbit IgG−PS−ALP can conjugate lots of ALP molecules (103−105 ALP per PS32) because of its large surfaceto-volume ratio, thus significantly enhancing the efficiency of bioluminescent reaction. Moreover, the ABS employs nanoparticles (MNPs and PS) to construct a homogeneous sensing strategy, dramatically shortening the time of the one-step immunoreaction within 30 min. In contrast, conventional ELISA is a heterogeneous method that needs multiple reactions and washing steps, and the whole analysis time is 4−6 h. More importantly, the bioluminescent signal in the ABS is quantitatively detected by a portable ATP detector within 10 s, while conventional ELISA requires a bulky instrument for readout. The ABS with improved sensitivity, simplified operation, and straightforward readout is thus well-suited for POCT of biomarkers. Detection of PCT. We next employ the ABS to detect PCT, an important biomarker for bacterial infection. The clinically relevant concentration of PCT in normal human serum is very low (0−50 pg/mL). Its concentration increases when the body suffers from bacterial infection, and the concentration of PCT in serum can reflect the degree of bacterial infection. To detect PCT of trace amount in serum (the clinical cutoff of PCT is 100 pg/mL), Ab1 (anti-PCT capture monoclonal antibody)− MNPs and Ab2 (anti-PCT detection monoclonal antibody)− PS−ALP are used to construct the ABS. For PCT ranging from 0.05 to 105 pg/mL, the ΔBI accordingly increases (Figure 5A), and a linear relationship between ΔBI and the concentration of PCT ranges between 1 and 104 pg/mL (Figure 5B). The LOD of the ABS for detection of PCT is 0.045 pg/mL (∼3.5 fM of PCT). We also investigate the performance of conventional

Figure 5. Comparison between the ABS, conventional ELISA, and Roche-ECL for detection of PCT. (A) The standard curve of the ABS and conventional ELISA for PCT detection in PBS solution (30% fetal calf serum). The concentration of PCT is from 0 to 105 pg/mL. (B) The linear range of the ABS and conventional ELISA for PCT detection (PCT ranging from 1 to 104 pg/mL). (C) The results of the ABS and conventional ELISA for PCT detection in real serum samples. Samples 1−5 are negative samples, and samples 6−20 are positive samples. The dashed red line presents the cutoff value (100 pg/mL) for PCT detection. (D) The comparison of PCT levels measured by the ABS and the Roche-ECL; a good consistency between the two methods is observed.

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CONCLUSION In this work, we employ the double-enzymes-mediated bioluminescent reaction, conjugated nanoparticles, and portable ATP detector to construct a rapid, highly sensitive, and 5426

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quantitative POCT assay (ABS). The ABS is applied for detection of trace amounts of PCT in real clinical samples and shows good consistency with the Roche-ECL relying on the expensive and bulky instrument. The ABS with features of high sensitivity, straightforward signal readout, and convenient operation may become an attractive POCT platform in biochemical analysis.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b00239. Additional experimental data, including the sensitivity of the glucose meter for detection of glucose, the stability of the ABS, the determination of PCT in real samples by the ABS and ELISA, the determination of PCT in real samples by the ABS and Roche-ECL, the process of portable ATP detector to measure the bioluminescent signal in the ABS, optimization of conditions of the ABS for detection of ALP, and the selectivity of the ABS for detection of PCT (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *Phone: 86 10 8254 5558. Fax: 86 10 8254 5631. E-mail: [email protected]. ORCID

Xingyu Jiang: 0000-0002-5008-4703 Author Contributions §

Y.C., Y.X., and J.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by NSFC (81671784, 21505027, 21475028, 21622503, 31622025 and 81361140345) and MOST (2013AA032204).

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REFERENCES

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DOI: 10.1021/acs.analchem.7b00239 Anal. Chem. 2017, 89, 5422−5427