Mycobacterium tuberculosis strain H37Rv Electrochemical Sensor

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M. tuberculosis strain H37Rv electrochemical sensor mediated by aptamer and AuNPs-DNA Xiao Qing Zhang, Ye Feng, Shaoyun Duan, Lingling Su, Jialin Zhang, and Fengjiao He ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01230 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019

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M. tuberculosis strain H37Rv electrochemical sensor mediated by aptamer and AuNPs-DNA Xiaoqing Zhang †,‡, Ye Feng†, Shaoyun Duan†, Lingling Su†, Jialin Zhang†, Fengjiao He*,† † State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China, 410082 ‡ School of Pharmacy, Hunan University of Chinese Medicine, Changsha, China, 410208 KEYWORDS H37Rv, MSPQC, aptamer, AuNPs-DNA, electrochemical sensor, Mycobacterium tuberculosis, SELEX, AuIDE ABSTRACT: The accurate and rapid detection of Mycobacterium tuberculosis (M. tuberculosis) is essential for the effective treatment of tuberculosis. In this article, we propose an electrochemical sensor to detect M. tuberculosis reference strain H37Rv. The sensor contains an H37Rv aptamer and oligonucleotides modified with gold nanoparticles (AuNPs-DNA). An H37Rv aptamer screened by our laboratory was used as the recognition probe. The change in frequency shift mediated by AuNPs-DNA in the presence of H37Rv was detected using a multichannel series piezoelectric quartz crystal (MSPQC) system. Three oligonucleotides modified with gold nanoparticles were designed. These oligonucleotides contained 12, 12, and 13 bases that hybridized with the 37-nt H37Rv aptamer. H37Rv aptamer was immobilized on the gold electrode surface by Au-S bonds. A conductivelayer was then formed by sequential hybridization of the aptamer with the three designed AuNPs-DNAs. When H37Rv was present, it specifically bound to the aptamer, resulting in the detachment of AuNPs-DNA from the electrode. The conductive layer was thereby replaced by a non-conductive complex of aptamer and bacteria. These changes were monitored by the MSPQC system. The proposed sensor is rapid, specific and sensitive, the detection time was 2 h. The limit of detection limit was 100 cfu/mL. This sensor would be of great benefit for the early clinical diagnosis of tuberculosis.

Tuberculosis (TB) is an airborne infectious disease caused by M. tuberculosis.1 It has killed millions of people and remains one of the most important infectious diseases worldwide. According to the World Health Organization, 1.7 million people died of tuberculosis and there were 10.4 million new cases worldwide in 2016.2 One of the key requirements to decrease the hazard of TB is rapid detection.3 Traditional TB detection methods, including bacterial cultivation assay,4,5 Ziehl-Neelsen staining microscopy,6 Mantoux tests7 and chest radiology8 are time-consuming, labor-intensive or imprecise9, thus cannot meet diagnostic needs. Some molecular assays, such as GeneXpert-PCR10, 11 and surface plasmon resonance (SPR),12-14 have been developed using M. tuberculosis DNA as a target, but these require preparation of DNA samples. PCR, including cell lysis and cycle amplification, usually takes 3–5 h. The application of SPR, with harsh experimental conditions and high costs, is limited in developing countries. The difficulty of isolation of monoclonal antibodies and costly processing seriously restrict the application of immunoassay methods.15 Therefore, in the global fight against TB, development of an economic, simple, rapid and effective detection method for the virulent M. tuberculosis strain H37Rv remains a key issue.16 Interdigital electrode piezoelectric quartz crystal (IDE-PQC) sensors are widely used in the biosensing due to their fast-response, high-sensitivity and low-cost.17 Our research team designed a series of MSPQC systems that can sensitively respond to changes in electrical parameters. A series of sensors based on the MSPQC system has been designed for the detection of M. tuberculosis.

These sensors include an immunological assay (which was expensive and based on antigen–antibody reaction),18,19 the aptamer sensor method (which needs the presence of the filtrate protein CFP10-ESAT6 after 96 h of incubation),20 the culture method (which was based on the detection of small molecular products of metabolism)21 and the phage amplification method (which also depended on cultivation, the detection time was reduced to 30 h).22 However, these detection methods relied on the cultivation of H37Rv, and cannot meet the needs of rapid detection. Aptamers are single-strand DNA or RNA oligonucleotides which were identified by the systematic evolution of ligands by exponential enrichment in vitro selection technique.23,24 They exhibit high specificity and affinity to their targets, low molecular weight, high reproducibility, easy chemical synthesis and modification, good stability, and reusability at room temperature. These advantages can overcome the restrictions of antibodies use in detection methods,25,26 and aptamers are thus widely used in biosensor.27 The H37Rv aptamer has been screened out by our research team and was designed to conjugate with single walled carbon nanotubes (SWCNTs) for the detection of H37Rv.28 However, it is not easy to form aptamer–SWCNTs conjugates along the electrons transport skeleton of SWCNTs, and ensure electron transport. Thus, the sensitivity of detection was limited in this method. In this paper, an AuNPs-DNA/H37Rv aptamer/MSPQC sensor was constructed for the rapid and sensitive detection of M. tuberculosis strain H37Rv. The high affinity sequence of the H37Rv

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Scheme

1.

The

schematic

of

the

detection

mechanism

aptamer was attached to an electrode as a recognition probe. Three DNA fragments attached on AuNPs hybridized sequentially with the H37Rv aptamer to form a conductive layer to enhance sensitivity. H37Rv bacteria then displaced these fragments from the aptamer, dramatically changing the conductivity. Thus, the sensor can rapidly detect H37Rv without cultivation and special labeling. The limit of detection (LOD) was 100 cfu/mL, and the detection time was 2 h. In addition, the proposed sensor was used to detect H37Rv in 40 processed clinical sputum samples, and the results showed no significant difference compared to the conventional detection method. EXPERIMENTAL SECTION Bacteria and Reagents. H37Rv (ATCC27294) was purchased from the National Institute for the Control of Pharmaceutical and Products. Mycobacterium smegmatis (M. smegmatis) and Bacillus Calmette-Guerin (BCG) were purchased from the Institute of Microbiology, Chinese Academy of Sciences. Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella enteritidis (S. enteritidis), and Pseudomonas aeruginosa (P. aeruginosa) were obtained from the National Institute for the Control of Pharmaceutical and Biological Production. (2carboxyethyl) phosphine hydrochloride (TCEP), Hydrogen tetrarchloroaura–te trihydrate (HAuCl4 3H2O), Sodium borohydride were purchased from Aladdin Biotech CO. Ltd. (Shanghai, China), 6-Mercapto-1-hexanol (MCH) was obtained from Sigma Aldrich (St. Louis, USA). All reagents were analytical reagent grade. SDS-PAGE gels were purchased from Sangon Biotech. Co., Ltd. (Shanghai, China) Sequences. The following sequences were synthesized by Shanghai Sangon Biological Engineering Technology and Services, Co., Ltd. (Shanghai,China) and used without further purification. H37Rv aptamer: 5´ACCCTGCGGGGCTGCCCGATATGTGTCCAAGTGGTGT-SH3´; Three DNA fragments: 5´-ACACCACTTGGA-C6- SH-3´, 5´CACATATCGGGC-C6-SH-3´ and 5´-AGCCCCGCAGGGT-C6SH-3´. Detection Medium. The medium composed of sodium hydrogen phosphate (2.5 g),ammonium sulfate (0.5 g), magnesium sulfate (0.05 g), calcium chloride(0.0005 g) copper sulfate (0.001 g), potassium dihydrogen phosphate (1 g), sodium citrate (0.1 g), zinc sulfate (0.001 g), pyridoxine hydrochloride (0.001 g), sodium glutamate (0.5 g), ferric ammonium citrate (0.04 g), biotin(0.0005 g), Tween-80 (0.5 g) in 1 L of distilled water, pH 7.2, and was separated into 95 mL, autoclaved at

for

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H37Rv

by

AuNPs-DNA/H37Rv-aptamer/MSPQC

sensor

121 °C for 20 min. Then, 5 mL of 10% oleic acid-dextrosecatalase were added to the bottles. All reagents were analytical pure and purchased from China Chemical Network (Changsha, China) Apparatus. HP4192A impedance analyzer (Hewlett Packard, USA). High-resolution transmission electron microscope JEM3010 (Japan Electronics Corporation, Japan) The MSPQC system was made in our laboratory (see Figure S1) Preparation of AuNPs. AuNPs (4 and 13 nm in diameter) were synthesized according to a previously reported method.29 The reaction vessel was thoroughly washed with freshly prepared aqua regia (HNO3/HCl = 1:3), and cleaned with double distilled water. Briefly, 200 µL of 1 mmol/L chloroauric acid was added to 20 mL ultrapurified water and quickly brought to the boil. Immediately, 0.72 mL of a 1% sodium citrate solution was added to synthesize gold nanoparticles with an average diameter of 13 nm. When the color of the solution changed from pale yellow to wine red, the AuNPs were formed. Similarly, 0.5 mL of 100 mmol/L chloroauric acid was added to 18 mL ultrapurified water, followed by 0.5 mL of 100 mmol/L sodium citrate at room temperature while stirring for 5 min. To generate particles with an average diameter of 4 nm, 0.85 mL of 0.1 M sodium borohydride was rapidly added to the above reaction solution, and the solution was allowed to react until its color changed from saffron yellow to orange red. AuNPs were formed and stored in the dark at 4 °C. The average sizes of AuNPs were estimated by transmission electron microscopy as shown in Figure S3. Preparation of AuNPs-DNA Bioconjugates. Preparation of AuNPs-DNA and their modification on the electrode: according to the method reported in the literature,30 the thiolaed DNA fragments were activated by 100-fold TCEP and reacted at room temperature for 1 h before loading onto the surface of AuNPs. Then, the mixture of 15 µL of 100 µmol/L DNA fragments and 500 µL AuNPs solution was mixed for 12 h at 4 °C. After incubation, 10 µL of 1% SDS was added, and the mixture was shaken for 1 h at room temperature to stabilize the AuNPs. Then 50 µL of 0.5 mol/L NaCl was added slowly and the mixture was incubated for 12 h. To remove unhybridized DNA fragments, the solution was centrifuged at 15000 rpm for 30 min, and the orange-red AuNPs-DNA prcipitate was washed three times, in 0.01 mol/L phosphate buffer (pH 7.4) with centrifugation and then suspended in 0.5 mL 2×SSC hybridization buffer (sodium citrate 30 mM, 300 mM NaCl) for future use.

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Figure 1. Gel electrophoresis (lane 1: 5 μL, 20 μM H37Rv aptamer sequence; lane 2: 5 μL, 20 μM of each of the three DNA fragments + 5 μL, 20 μM H37Rv aptamer sequence; lane 3: recovery solution of hybridization band in lane 2 + 5 mL, 1×10 7 cfu/mL of H37Rv). Modification of Electrode with H37RV Aptamer. The bare Au interdigital electrode (Au-IDE) was washed for 5 s with piranha solution (3:1 [V: V] H2SO4/30% H2O2), followed by ultrasonic cleaning with ethanol for 5 minutes, and then rinsed with double steamed water, and dried with N2. To produce a self-assembled H37Rv aptamer monolayer on the AuIDE, 10µL, 1 µM thiolated H37Rv aptamers solution (in 1 mM EDTA and10 mM Tris–HCl, pH 8.0) were incubated on the gold electrode overnight at 4 °C (the disulfide bond at the 3ʹend of H37Rv aptamer was previously cleaved with 100 times the amount of TCEP at room temperature for 1 h). Then 10µL, 1 mM MCH solution was dropped on the electrode surface and kept at room temperature for 1 h to block active site. Preparation of AuNPs-DNA/H37Rv Aptamer on the Electrode. Hybridization was carried out sequentially. AuNPsDNA solution (10μL in 2× SSC hybridization buffer) containing three different DNA fragments, respectively, was incubated on the electrode surface for 2 h at 37 °C. After each step, the electrode was rinsed with washing buffer (20 mM Tris-HCl, 0.1 M NaCl, 5 mM MgCl2) and dried with nitrogen. Gel Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 15%) was performed using 0.1× TBE as the running buffer. Each sample was loaded (lane 1: 5 μL, 20 μM H37Rv aptamer sequence. lane 2: three DNA fragments, each was 5 μL, 20 μM + 5 μL, 20 μM H37Rv aptamer; lane 3: recovery solution of hybridization band in lane 2 + 5 mL 1×107 cfu/mL H37Rv, resulting in the H37Rv aptamer– H37Rv complex) and the gel was run at a constant potential of 120 V for 60 min. Gels were photo-graphed by gel image system after Stains-All staining with GenGreen/GenRed nucleic acid dye solution for 30 min. Sample Detection. Test sample treatment: the concentration of standard strains was diluted to 0.5 McFarland units by McFarland turbidity meter (108 cfu/mL) and H37Rv was diluted to 102–107 cfu/mL with sterile saline. The clinical samples were placed in a sealed reagent tube and heated in a water bath at 85 °C for 30 min for inaction. The inactivated samples were added to the same volume of 10% NaOH solution. The samples were mixed by a vortex oscillator and liquefied at room temperature for 30 min. Liquefied sample (1 mL) was centrifuged for 5 min at 8000 rpm to remove the

Figure 2. Nyquist plots of impedance spectra obtained using the different electrode modified with (a) bare Au-IDE. (b) H37Rv aptamer/Au-IDE. (c) AuNPs-DNA/H37Rv aptamer/Au-IDE, where the H37Rv aptamer hybridized with DNA. (d) the H37Rv/H37Rv aptamer/Au-IDE, where AuNPs-DNA were replaced by H37Rv. supernatant. The sediment was washed three times with 1 mL sterile phosphate buffer (pH 6.8), and the final sediment was mixed with 1 mL detection medium. The detection sample solution was then ready for use. All the reagents were analytically pure. A total of 1 mL of treated samples solution was added to 4 mL of detection medium. Additionally, 1 mL H37Rv solution was added to 4 mL of detection medium as a positive control, and 5 mL of detection medium without H37Rv was used as the negative control. Frequency response curves were recorded using the MSPQC system. All samples were confirmed by acid-fast smear analysis. All experiments described here were operated in class II biological safety cabinets, and all experimental wastes and utensils were washed after autoclaving. RESULTS AND DISCUSSION Design of the Proposed Biosensor and the Response Mechanism. The design of the proposed gold nanoparticles AuNPs-DNA/H37Rv aptamer/MSPQC sensor is shown in Scheme 1. The recognition probe comprised an H37Rv aptamer layer modified with Au–S bonds on the Au-IDE of the MSPQC system. Redundant sites were blocked using MCH. The sequence and secondary structure of the aptamer are shown in supplementary Figure S2. DNA duplexes in the range of 5-20 bp were of conductive.31 Consequently, oligonucleotides of 12, 12, and 13 nt were attached to AuNPs and were designed to hybridize with H37Rv aptamer sequentially. A conductive layer was thereby obtained. When present, H37Rv specifically binds to the aptamer, resulting in the detachment of AuNPsDNA from the electrode. The AuNPs-DNA is replaced by nonconducting H37Rv, leading to charge transmission blockage of the electrode surface. As a result, the electrical parameters of the sensor system change. The MSPQC responds sensitively to the change, thereby enabling the proposed sensor to rapidly detect H37Rv. The MSPQC system developed by our laboratory is based on a quartz crystal oscillator circuit. This system provides a sensitive real-time, online response, simple operation, rapid detection, high sensitivity and low cost. The equivalent circuit of MSPQC system consists of three parts (as shown in Figure S4a): box I is the equivalent circuit model of the piezoelectric quartz crystal, box II is the equivalent circuit of the modified layer on the electrode,

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Table 1. Evaluation of AuNPs-DNA/H37Rv aptamer/MSPQC sensor by Culture Method proposed

Culture Method total

sensor

Figure 3. The typical response curves of (a) the proposed sensor in detection medium without H37Rv. (b) control experiments of the sensor using only the aptamer, without the AuNPs-DNA, in detection medium containing 1×107 cfu/mL H37Rv. (c) control experiments of the sensor using only three short DNAs, without the AuNPs, in detection medium containing 1×107 cfu/mL H37Rv. (d) the proposed sensor in detection medium containing 1×107 cfu/mL H37Rv.

Figure 4. The specific response to H37Rv compared with other bacterial strains. The concentration for all bacteria was 107 cfu/mL. Error bars indicate standard deviation (n=3). and box III is the equivalent circuit of the test solution. C0, Lq, Cq and Rq are static capacitance, dynamic inductance, dynamic capacitance and dynamic resistance of the piezoelectric crystal, respectively. Cf and Rf are the membrane capacitance and the film resistance respectively. The frequency shift was detected by the MSPQC system in accordance with the following equation:32

F  K1  Rt  K 2  Ct

(1)

K1 

  A  4 2 F02Ct2 Rt2 A  4 F0Ct Rt F     F02Cq   2 2 2 2 Rt  1  2 F0C0 Rt A  4 F0 Rt Ct (C0  Ct )   (2)

K2 

  1  4 2 F02Ct2 Rt2  4 A F0Ct Rt F    2 2 F03Cq   2 2 2 2 Ct  1  2 F0C0 Rt A  4 F0 Rt Ct (C0  Ct )   (3)

According to Eq. (1), the frequency shift of the proposed sensor is affected not only by the resistance of the medium solution, but also by its capacitance. The values of Rt and Ct are affected only by the electrical properties of the surface modifi

+

-

+

10

3

13

-

1

26

27

total

11

29

40

cation layer of the electrode in the high-conductivity detection medium. Therefore, the change of the electrical properties of the surface modification layer of the electrode causes the sensitive response of the AuNPs-DNA/H37Rv aptamer/MSPQC sensor. The Reaction of H37Rv Aptamers with AuNPs-DNA and H37Rv. To prove the occurrence of the hybridization between H37Rv aptamer and the three DNA fragments, and the replacement of the three DNA fragments in the hybridization chain by H37Rv, PAGE was performed. The results are shown in Figure 1. Lanes 1, 2, and 3 represent H37Rv aptamer, H37Rv aptamer hybridized with three different DNA fragments, and H37Rv aptamer–H37Rv complex, respectively. There was one light band and one darker band in lane 2. The brighter band indicated that the three DNA fragments had been successfully hybridized with H37Rv aptamer, and the darker band showed the residue of the unhybridized H37Rv aptamer. However, the DNA fragments not hybridized with H37Rv aptamer had run out of the PAGE gel because of their short length and high electrophoresis speed. The band at the notch in lane 3 demonstrated that H37Rv successfully replaced AuNPs-DNA and formed H37Rv aptamer–H37Rv complex. The complex did not migrate during gel electrophoresis because of the presence of bacteria. These results validated our probe design. Electrochemical Impedance Characterization and Typical Response Curve. The impedance characteristics of the electrode were investigated using an HP-4192A LF impedance analyzer, as shown in Figure 2. Curve (a) shows the impedance characterization of the bare Au-IDE. The diameter of the semicircle in curve (b) was larger than that in curve (a), thereby indicating that the H37Rv aptamer used as the recognition probe had been successfully attached to the Au-IDE by Au-S bonds. The impedance increased because the self-assembled monolayer of H37Rv aptamer blocked the charge transfer. For curve (c), the diameter of the semicircle was markedly smaller than that in curve (b), indicating that the AuNPs-DNA had successfully hybridized with H37Rv aptamer and formed the conductive layer on the surface of Au-IDE. This layer significantly enhanced the charge transfer on Au-IDE surface. The diameter of the semicircle in curve (d) was much larger than that in curve (c), indicating that AuNPs-DNA had been successfully displaced by H37Rv, thereby forming a nonconductive complex layer on the surface of Au-IDE. The AuNPs-DNA/H37Rv aptamer/MSPQC sensor, constructed in accordance with the above-described design, was used to detect 1×107 cfu/mL H37Rv. A typical response is shown in curve (d) of Figure 3. Curve (b) shows that when H37Rv was present in the sample solution, H37Rv aptamer combined with

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ACS Sensors

Figure 5. (A) Response curves of different concentration of H37Rv (a, 1×102 cfu/mL; b, 1×103 cfu/mL; c, 1×104 cfu/mL; d, 1×105 cfu/mL; e, 1×106 cfu/mL; f, 1×107 cfu/mL ); (B) The calibration curve between the frequency change and H37Rv concentration (logarithm). The measurements were repeated 3 times to obtain the standard deviation. H37Rv. As a result, AuNPs-DNA detached from the Au-IDE, and the conductive layer formed by AuNPs and short-stranded DNA was replaced by nonconductive bacteria. Consequently, the charge transfer on the electrode surface was blocked, and this resulted in a significant increase in the impedance value. According to Eq. (1), the detection curve reached a steady state with the completion of the replacement of AuNPs-DNA and H37Rv, thereby indicating the stability of H37Rv aptamer-H37Rv complex on the electrode surface. The results could be judged in 60 min. If H37Rv was not present in the sample solution, the conductive layer formed by H37Rv aptamer and AuNPs-DNA on the electrode surface remained stable, and the oscillation frequency did not change, which is shown in curve (a). Therefore, the change of frequency shift can determine the presence of H37Rv. The experimental results showed that the frequency shift caused by factors other than the presence of H37Rv did not exceed 30 Hz. Therefore, positive results can be reported when the frequency shift value is >30 Hz. However, when the probe contained aptamers alone, i.e., without AuNPs-DNA, the frequency shift was very small, as shown in curve (b). This was because the electron transport of aptamers and bacteria was very poor. In the same way, if the three short oligonucleotides without AuNPs were used to hybridize with the aptamer to form a rigid double-strand, the resulting frequency shift, as shown in curve (c), was small. This was because, when the three short oligonucleotides were replaced by bacteria, and H37Rv–aptamer complex was formed, the charge transfer capability of the electrode surface was not enhanced. Effect on the Response Signal of the H37Rv Aptamer Concentration Used for Probe Construction. The effect on the response to H37Rv (1 × 106 cfu/mL) of the concentration of H37Rv aptamer used in the proposed sensor was investigated. The results are shown in Figure S5. H37Rv aptamer (10μL) at concentrations of 0.5, 1, 2, 4, and 6 μmol/L was used to modify the Au-IDE of MSPQC, and a set of AuNPs-DNA/H37Rv aptamer/MSPQC sensors was prepared. The detection time was 2 h and three parallel experiments were carried out for each sensor type. When the concentration of H37Rv aptamer used in the preparation of the sensor was 1 mol/L, the frequency shift was the highest. For the H37Rv aptamer at 2 mol/L and 4 mol/L, the frequency shift were slightly lower than that for 1 mol/L. The frequency shift for 0.5 mol/L and 6 mol/L H37Rv aptamer were lower than those for the other concentrations. When the concentration was increased to 6 mol/L, the hybridization efficiency between the aptamer and

AuNPs- DNA was affected by the high concentration, and this, in turn, affected the resulting frequency shift. Therefore, the concentration of H37Rv aptamer used in the proposed sensors in all subsequent experiments was 1 mol/L. Effect of AuNPs Particle Size on the Response Signal. To investigate the effect of AuNPs size on the sensor response signal, AuNPs-DNA were prepared using AuNPs with two average diameters: 4 and 13 nm. The frequency shifts of the sensors obtained in three parallel experiments in the presence of H37Rv (1 × 106 cfu/mL) were 167 and 100 Hz, repecttively. The reason for the higher frequency shift signal of 4 nm AuNPs may be their large radius of curvature and small steric hindrance, which would facilitate dehybridization between AuNPs-DNA and H37Rv aptamer,33 and promote the replacement of AuNPs-DNA by H37Rv. Selectivity of the AuNPs-DNA/H37Rv Aptamer/MSPQC Sensor. To evaluate the selectivity toward H37Rv of the proposed sensor, a series of potential interfering bacteria were used at concentration of 1 × 107 cfu/mL. The potentially interfering bacteria used were E. coli, BCG, M. smegmatis, P. aeruginosa and S. aureus; a sterile test solution was used as a blank control. The detection results are shown in Figure 4. The ΔF value of H37Rv was 236 Hz, whereas the ΔF values of the other bacteria were no more than 25 Hz. This showed that many types of experimental bacteria [including cocci (e.g., S. aureus) and bacilli (e.g.,P. aeruginosa, E. coli)] did not interfere with H37Rv detection. In particular, neither M. smegmatis (a nonpathogenic mycobacteria) nor BCG interfered with H37Rv detection. Therefore, the proposed sensor can distinguish pathogenic from non-pathogenic bacteria. Influence of H37Rv Concentration on the Response Signal and the Detection Limit of the Sensor. Different concentrations of H37Rv were detected using the proposed sensor. The response curves are shown in Figure 5A. The frequency shift increased with the increase of H37Rv concentration. Large amounts of AuNPs-DNA complex detached from the surface of electrode because of the specific reaction between H37Rv aptamer and H37Rv. The conductive layer on the surface of the electrode was, consequently, replaced by a nonconductive layer. As a result, ΔF increased with the increase of Rt. A linear relationship between the logarithmic values of H37Rv concentration and the frequency shift was found as the H37Rv concentra tions changed from 1 × 102 – 1 × 107 cfu/mL (Figure 5B). The equation ΔF = 34.57 logc – 33.12 (R2=0.9375) was obtained. The standard deviation of three

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Table 2 Comparison of AuNPs-DNA/H37Rv aptamer/MSPQC sensor method with other previously reported method for detection of M. tuberculosis Method

LOD, cfu/mL

Reponse target

Detection time (h)

Specificity

Refs.

Traditional culture

10

Count colony

720~1440

No

(34),(35)

BACTEC MGIT 960

sensitivity of 81.5%

O2

168 -240

No

(36)

MSPQC

10

NH3+CO2

48~192

No

(20)

SPR

1×104

Ag85 protein

0.05), there was no significant difference between the two methods. The proposed sensor was evaluated by using the results of L-J slant culture method. The sensitivity was 91% (10/11), and the specificity was 90% (26/29), as shown in Table 1. However, the average detection times for the AuNPsDNA/H37Rv aptamer /MSPQC sensor and L-J slant culturemethod were 60 min and 21 days. These results demonstrated that the proposed method was accurate, rapid, and simple. Comparison between the current AuNPs-DNA/H37Rv Aptamer/MSPQC Sensor and other Detection Methods. A comparison of the sensor proposed here with other reported methods for detection of M. tuberculosis is shown in Table 2. The detection target of the proposed method was whole H37Rv cells, which do not require culture and extraction. However, other methods used DNA fragments; secreted proteins Ag85, ESAT-6, or CFP-10; or culture metabolites as targets for detecting H37Rv. These other methods require the training of personnel and complex extraction operations. Moreover, the proposed MSPQC-based sensor can sensitively and quickly capture changes in electrical signals. The proposed method was therefore shown to be sensitive, simple, and rapid. CONCLUSIONS

ASSOCIATED CONTENT The structure of MSPQC system; the secondary structures of H37Rv aptamer; TEM image and UV–vis absorption spectra of AuNPs-DNA and AuNPs; equivalent electric circuit of proposed sensor; the effect of H37Rv aptamer concentration on the response signal ΔF.This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Present Addresses †If an author’s address is different than the one given in the affiliation line, this information may be included here.

Funding Sources The National Natural Science Foundation of China (No. 21275042); Project supported by the Research Foundation of Education Bureau of Hunan Province, China (No. 14C0860)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 21275042) and A Project Supported by Scientific Research Found of Hunan Province Education Department, China (No. 14C0860) and Department of Clinical Laboratory of Tuberculosis Hospital of Hunan Province.

The proposed AuNPs-DNA/H37Rv aptamer/MSPQC sensor based on H37Rv aptamer, AuNPs-DNA, and a sensitive MSPQC detetion system was a rapid, simple, sensitive, and specific method for detecting Mycobacterium tuberculosis. Using the proposed method without culturing, the detection time of strain H37Rv was 2 h.

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