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Ultrasensitive detection of Shigella species in blood and stool Jieling Luo, Jiapeng Wang, Anup S. Mathew, and Siu-Tung Yau Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04242 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 17, 2016

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Analytical Chemistry

Ultrasensitive detection of Shigella species in blood and stool

Jieling Luo1, Jiapeng Wang1, Anup S. Mathew1 and Siu-Tung Yau1,2*

1. Department of Electrical and Computer Engineering, Cleveland State University, Cleveland, Ohio 44115, USA

2. The Applied Bioengineering Program, Cleveland State University, Cleveland, Ohio 44115, USA

* Corresponding author. E-mail, [email protected]; phone, (216) 875-9602; fax, (216) 678-5405

Abstract A modified immunosensing system with voltage-controlled signal amplification was used to detect Shigella in stool and blood matrices on the single-digit CFU level. Inactivated Shigella was spiked in these matrices and detected directly. The detection was completed in 78 min. Detection limits of 21 CFU/mL and 18 CFU/mL were achieved in stool and blood, respectively, corresponding to 2-7 CFUs immobilized on the detecting electrode. The outcome of the detection on extremely low concentrations, i.e. below 100 CFU/mL, blood samples shows a random nature. An analysis of the detection probabilities indicates the correlation between the sample volume and the success of detection and suggests that sample volume is critical for ultrasensitive detection of bacteria. The calculated detection limit is qualitatively in agreement with the empirically determined detection limit. The demonstrated ultrasensitive detection of Shigella on the single-digit CFU level suggests the feasibility of direct detection of the bacterium in samples without performing culture.

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Introduction Early detection of infectious bacteria is an indispensable component of strategies for combating infectious diseases1. Containing the spread of infectious diseases requires effective monitoring for signs of an outbreak and rapid diagnosis of its microbial cause. Clinically, rapid detection of small numbers of infectious bacteria has profound consequences in the early diagnosis and effective treatment of infectious diseases.

Current methods mostly used for the detection of bacteria are

polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), both of which, depending on the bacteria concentration, require an 7-24 hour culture-based enrichment of samples to bring the number of bacteria to a detectable level2.

For the diagnosis of bacteremia or

bloodstream infections, culture is used to detect bacteria in blood at the level of 10 CFU/mL3. The culture process requires at least 48 hours, during which wide-spectrum antibiotics are prescribed for temporary treatment. However, overuse of broad-spectrum antibiotics leads to the prevalence of drug-resistant organisms4. To significantly shorten the diagnosis, ultrasensitive detection methods for bacteria are needed to detect extremely small numbers of bacteria directly without culturing the sample.

Shigella is a genus of Gram-negative, rod-shaped bacteria. It is the causative agent of shigellosis, an infectious disease with symptoms of bloody diarrhea, fever, and stomach cramps. Shigellosis is diagnosed through laboratory testing of stool samples for the presence of Shigella cells. Shigellosis may leads to bacteremia5. In this article, we show that a modified immunosensing system that provides voltage-controlled intrinsic signal amplification is able to detect inactivated Shigella directly in stool and blood at the single-digit CFU level. The detection in stool samples is characterized by a wide linear range from 50 to 3x104 CFU/mL. The performance of the detecting electrodes appears to be stable over a period of 21 days. The detection of Shigella in whole human blood shows a random nature below 100 CFU/mL. Between 20-70 CFU/mL, the probability of a 2 ACS Paragon Plus Environment

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successful detection improves as the sample volume is increased. Each detection was completed in 78 min. The fact that the detection system is able to provide ultrasensitive detection of extremely small amounts of bacteria due to the signal amplification suggests the feasibility of direct detection of the bacterium in samples without performing culture, leading to rapid detection of bacteremia. In the present work, Shigella was used to demonstrate the potential of the detection method in early detection of infectious diseases, especially in the detection of bacteremia without culturing the sample.

Experimental Section Experimental system Figure 1 describes the detection system, which is a modified three-electrode electrochemical cell with a cell potential Vcell connected between the working electrode and the reference electrode. The cell contains phosphate buffer saline (PBS) and is driven by an electrochemical potentiostat (not shown). The cell is modified with insulated gating electrodes for applying a gating voltage VG between the gating electrode and the working electrode, upon which horseradish peroxidase (HRP), a redox enzyme, is immobilized via the sandwich immune complex. VG modifies the charge distribution at the solution-enzyme-electrode interface to induce an electric field which penetrates the immune complex to reduce its tunnel barrier (height of potential energy profile). Therefore, the tunnel current between the electrode and HRP can be amplified. A detailed description of the principle of the system is available in a previous publication6 and in Supporting Information. Reagents Inactivated (heat-killed) Shigella (positive control) and antibodies specific to Shigella were purchased from KPL (Gaithersburg, MD; product number 50-90-01). The positive control, based on disease prevalence, consisted of a mixture of Shigella boydii, flexneri, and dysenteriae.

The

CFU/mL of the Shigella positive control was estimated by counting live CFU on Agar plates prior to 3 ACS Paragon Plus Environment


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heat treatment. Polyclonal antibody with a known concentration was produced by inoculating the positive control to rabbits. After its production, the antibodies in antisera were selected with whole, live Shigella using the Encapsulated Column Affinity Purification Technology. Polyclonal antibody was used as the capture antibody and HRP-conjugated polyclonal antibody was used as the detection antibody. Artificially synthesized stool was purchased from ClaremontBio Solutions (Upland, CA). Human blood was purchased from Valley Biomedical (Winchester, Virginia). The stool and blood samples were used as negative controls. Shigella was spiked in the stool and blood samples as positive controls. De-ionized water (18 MΩ cm) was used to prepare PBS. All reagents were used as received without further processing. As a note, testing the detection system using the positive control demonstrates the detection capability of the detection device/system. Since the antibodies are selected using live bacteria, they capture live bacteria in samples in real applications.

Detecting electrodes Screen printed electrodes (SPEs) were used as the detection electrode for low-cost, disposable and near-patient use. Commercial SPEs were purchased from Metrohm (Riverview, FL). Its working electrode as shown in Figure 2 is a circular carbon electrode with a 4-mm diameter. The working electrode, silver reference electrode (RE) and the carbon counter electrode (CE) are fabricated on the top side of the SPE. The dimensions of the SPE are 3.4 cm x 1.0 cm x 0.05 cm.

SPEs were used as the basic detecting electrode. The carbon working electrode of the SPE was covered with a layer of polyaniline (PANI), upon which the capture antibody was immobilized via passive adsorption. Different concentrations of the capture antibody were used to maximize the detection signal. The orientation of the antibody was not controlled. A metallic wire wrapped with an insulator was used as the gating electrode. To construct the detecting electrode, the capture antibody was immobilized on the working electrode by an overnight incubation followed by washing 4 ACS Paragon Plus Environment

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the electrode with de-ionized (DI) water. The detection procedure consisted of a 35-45 min incubation of the working electrode with positive controls followed by washing electrode with DI water. Then, the electrode was incubated with a solution containing the detection antibody for 35 min followed by washing with DI. The sandwich immune complex now was formed on the working electrode of the SPE and ready for detection measurement. Operation of system The SPE was controlled by a commercial electrochemical potentiostat (CHI 660C Work Station). A piece of 1 mm-diameter copper wire coated with plastic was wound around the SPE and used as the gating electrode. The SPE-gating electrode cross-sectional structure is illustrated by Figure 1. The gating electrode was connected to the working electrode via a dc power supply to apply VG to the working electrode of the SPE. The SPE-gating electrode assembly was contained in a cell, which also contained PBS. Cyclic voltammograms (CV) were generated using the potentiostat. The reduction peak current of HRP shown in the CVs of the SPE was used as the detection signal. Since no diffusional mediators and H2O2, which is the substrate of HRP, were used in the operation of the detection system, the detection method embodies a reagent-less approach. Different values of VG between 0.1-0.6 V were tested. The amplification provided by VG=0.6 V was high enough for measurements.

The cross-reactivity of the system was tested by mixing different amounts of inactivated positive control of E. coli with a fixed amount of Shigella and detecting Shigella. It was found that for a given amount of E. coli, the Shigella detection signal became less than that measured in the absence of E. coli. The detection signal associated with the same amount of Shigella became further reduced with increasing amounts of E. coli. This observation suggests that E. coli first physically adsorbed to the binding sites of the capture antibody, blocking their access to Shigella. Since no immune-reaction 5 ACS Paragon Plus Environment


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was involved in this process, E. coli was subsequently removed from the electrode by the washing step. Therefore, the observation does not indicate cross-reactivity and hence the absence of falsepositivity. The loss of detection signal of Shigella can be remedied by increasing the electrode size. This method should work for other bacteria. Results and Discussion Verification of system operation The effect of VG on the detection signal was tested and verified with Shigella spiked in PBS and the results are shown in Figure 2. CV1 in Figure 3(a) was obtained with a SPE immobilized with 3x104 CFU/mL of Shigella without applying VG. The reduction peak of HRP is located near – 0.4 V vs. Ag/AgCl as previously noted7. CV2 was obtained with VG = 0.6 V. Comparing the peak height of CV1 with that of CV2 shows that VG causes the peak height to increase from 15µA to 46 µA, indicating signal amplification. As a note, the location of the peak remains near -0.4 V in CV2. Figure 3 (b) shows the effect of VG on low concentration samples. All of the three CVs were obtained with a SPE immobilized with 100 CFU/mL of Shigella. Without applying VG, a weak HRP reduction peak (the detection signal) is detected as indicated by CV1. With VG=0.6 V, a welldefined reduction peak with increased peak current appears as shown by CV2. CV3 shows that when VG is set back to 0 V, the HRP reduction peak returns to its original shape. When PBS is used as negative control, the CVs appear to be featureless with and without applying VG, indicating the absence of the immune complex on the electrode. The CVs were obtained in the presence and absence of the capture antibody. The CV with the antibody shows a slightly larger background current than that for the CV without. VG does not produce features on the CVs. The CVs are shown in Figure S3 in Supporting Information. Since no reagent was used in obtaining the HRP reduction peak, the detection method embodies a reagent-less detection approach.


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Detection of Shigella in stool Shigella was spiked in synthesized stool. The stool mixture consists of bile salts, dextran sulfate, human genomic DNA, mucin, human serum albumin and water. Detection of Shigella in stool was performed directly without further processing the sample. The calibration curve of Shigella in stool obtained with VG = 0.6 V is shown in Figure 4 (a). The detection measurements were performed with a sample volume of 50 µL taken from a 1 mL sample of the same concentration. The first five data points define a linear range. Higher concentration (3x103-3x104 CFU/mL) data points show a deviation from the linearity possibly due to less available binding sites or crowding effect. The calibration curve covers the range of 50-3x104 CFU/mL. Note that a completely linear calibration curve of an assay method, although desirable, is not mandatory for diagnosis of infectious diseases8, 9

. The red line in Figure 4 (a) is the regression line for the first five data points, having a correlation

coefficient of 0.866. The analytical sensitivity derived using the red line is 0.014 µA mL/CFU. The calculated detection limit of this Shigella concentration range is 21 CFU/mL, using the signal/noise=3 method.

Shigella has a low infectious dose of 10 to 100 CFU

10

. The linear range of the calibration curve

covers 50-1000 CFU/mL. Within this range, the number of CFU bound to the electrode via the immune complex corresponds to 3-50 CFU. Therefore, the detection system demonstrates the capability of detecting Shigella on a level below the infectious dose, indicating the potential of early detection of shigellosis.

The long term stability of the detecting electrode was characterized by storing detecting electrodes at 4°C for periods up to twenty one days and performing cyclic voltammetry with the electrodes. Figure 4 (b) shows three CVs, namely, CV1, CV2 and CV3, taken over periods of 7, 14 and 21 days, 7 ACS Paragon Plus Environment


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respectively. The electrodes used in these measurements were immobilized with 3x104 CFU/mL of Shigella in stool and the CVs were taken with VG = 0.6 V. Each of the three CVs shows a reduction peak of HRP. The peak currents are, starting from the 7-day measurement, 8.3 µA, 8.2 µA and 8.2 µA. The peak currents indicate that the performance of the detecting electrodes is stable over time.

Detection of Shigella in blood Shigella was also spiked in whole human blood. Whole blood is an extremely complex matrix as it contains platelets, white blood cells, red blood cells, and plasma together with hormones, enzymes, electrolytes, sugars, and other nutrients. Detection of Shigella was performed directly without further processing the sample. The detection measurements were performed with a sample volume of 50 µL taken from a 1 mL sample of the same concentration. The calibration curve of Shigella in blood obtained with VG = 0.6 V is shown in Figure 5 (a). The curve covers the range of 20-3x104 CFU/mL.

The first seven data points define a linear range. Higher concentration (3x103-3x104

CFU/mL) data points show a deviation from the linearity possibly due to less available binding sites or crowding. The red line is the regression line for the first seven data points, having a correlation coefficient of 0.828. The analytical sensitivity derived using the red line is 0.027 µA mL/CFU. The calculated detection limit of this concentration range is 18 CFU/mL, using the signal/noise=3 method.

In Figure 5 (a) each data points in the calibration curve between 100 and 3x104 CFU/mL was measured using three electrodes, each of which yielded a detection signal. Figure 5 (b) shows the details of the low concentration portion of the calibration curve, where, below 100 CFU/mL, the detection showed a random nature. Between 70 CFU/mL and 20 CFU/mL, not every single detection run resulted in a signal peak. Table 1 shows the statistical nature of the low-concentration detection 8 ACS Paragon Plus Environment

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measurements. The two rows of results show respectively the detection probabilities at different Shigella concentrations obtained using 50 µL and 100 µL as the volume of the measurement sample with both volumes taken from 1 mL sample of the same concentration. Within each box of the rows, the upper registry shows the probability of the success of detection (appearance of signal peak), and the lower registry shows the number of Shigella CFU nominally immobilized on the electrode. Table 1 shows that, below 100 CFU/mL, randomness in the success of detection occurs in the measurements in both rows. In the first row, the probability decreases from ¾ (0.75) for 70 CFU/mL to 4/7 (0.57) for 50 CFU/mL and then to 0/4 (0) for 20 CFU/mL. The decreasing probabilities are consistent with the decreasing bacteria concentrations. The second row shows that, for the same bacteria concentration, the probability of successful detection increases as the sample volume is increased from 50 µL to 100 µL. Therefore, Table 1 with the probabilities shown in the horizontal and vertical directions shows the effect of sample volume. When the concentration is extremely low, the volume of the sample used to make a detection measurement may not contain bacteria cells3. With reference to the high concentration data points obtained with unity probability, the absence of the signal peak in low concentration measurements is due to the difficulty in including bacteria cells in the measurement sample volume. Therefore, the randomness of the disappearance of the signal peak does not imply the incapability of the detection system to detect small amounts of cells.

In fact, the detection measurements made with low concentration samples as shown in Table 1 embody another method for the determination of the detection limit.

In the demonstration of

diagnostic assays, in addition to calculating the detection limit as shown above, the detection limit can be also empirically determined by testing serial dilutions of samples with a known concentration of the target substance in the analytical range of the expected detection limit11. In fact, the empirical method is more useful for infectious diseases diagnosis 12. Based on the results shown in Table 1 and the discussion given above, the detection limit of Shigella estimated using the empirical method 9 ACS Paragon Plus Environment


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should be below 50 CFU/mL, corresponding to the detection of 2.5 CFU or less of Shigella. Therefore, the empirically determined detection limit is qualitatively in agreement with the calculated one.

Conclusions This work shows that a modified immunosensing system, which provides voltage-controlled signal amplification, is capable of detecting inactivated Shigella species in stool and blood on the singledigit CFU level in 78 min. The detection of 2-7 CFUs of Shigella indicates that the detection system has the potential of detecting shigellosis in its early stage. In the study of the random nature of detection below 100 CFU/mL in blood, a correlation between the sample volume and the success of detection was observed, indicating the importance of sample volume with extremely low concentration samples. The ultrasensitive detection capability provided by the detection system suggests the feasibility of direct detection of the bacterium in samples without performing culture, leading to rapid detection of bacteremia. This work does not imply that it is a validation of a detection method. Rather, it only describes a new detection method that demonstrates the suitability for the diagnosis of bacteremia compared with existing bacteria detection methods. As a note, the mixture of the three Shigella species and the absence of S. sonnei in the positive control indicate the limitations in the detection of Shigella species due to the availability of the control sample. Detailed quantification of the cross-reactivity study reported here is will be carried out in future studies with the antibody titre and with different electrode size as parameters.

Acknowledgement The work was supported by Cleveland State University (Faculty Research and Development Award). JL acknowledges the support provided by the College of Graduate Studies at Cleveland State University during 2014-2015. 10 ACS Paragon Plus Environment

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Conflict of Interest Disclosure The authors declare no competing financial interest.

Supporting Information. The principle of field effect enzymatic detection; additional control measurements.

References (1)

Diekema, D. J.; Pfaller, M. A. Clinical Infectious Diseases 2013, 56, 1614-1620.

(2)

Lazcka, O.; Campo, F. J. D.; Munoz, F. X. Biosensors and Bioelectronics 2007, 22,

1205–1217. (3)

Weinstein, M. P. Clinical Infectious Diseases 1996, 23, 40-46.

(4)

Antibiotic Resistance Threats in the United States, 2013. Centers for Disease Control and

Prevention 2013. (5)

Grondin, C.; Imbert, P.; Ficko, C.; Merens, A.; Dutasta, F.; Bigaillon, C.; Rapp, C.

Journal of Travel Medicine 2012, 19, 258–260. (6)

Choi, Y.; Yau, S.-T. AIP Advances 2011, 1, 042175.

(7)

Ferri, T.; Poscia, A.; Santucci, R. Bioelectrochemistry and Bioenergetics 1998, 44, 177-

181. (8)

CLSI/NCCLS. 2003. Evaluation of the linearity of quantitative measurement procedures:

a statistical approach. Approved guideline. CLSI document EP2006-A. Clinical and Laboratory Standards Institute, Wayne, PA. (9)

Westgard, J. O. Basic method validation, 3rd ed ed.; Westgard QC, Inc.: Madison, WI.,

208. (10)

Warren, B. R.; Parish, M. E.; Schneider, K. R. Critical Reviews in Food Science and

Nutrition 2006, 46, 551–567.



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(11)

CLSI/NCCLS. 2004. Protocols for determination for limits of detection and limits of

quantitation. Approved guideline. CLSI document EP2017-A. Clinical and Laboratory Standards Institute, Wayne, PA. (12)

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Burd, E. M. Clinical Microbiology Reviews 2010, 23, 550–576.


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Table 1 Low concentration detection results

Concentration Volume

50µL

100µL

20 CFU/mL

50 CFU/mL

70 CFU/mL

100 CFU/mL

150 CFU/mL

0/4 (0)

4/7 (0.57)

3/4 (0.75)

3/3 (1)

3/3 (1)

1 CFU

2.5 CFU

3.5 CFU

5 CFU

7.5 CFU

1/4 (0.25)

5/7 (0.71)

4/5 (0.8)

2 CFU

5 CFU

7 CFU

Within each box of the rows, the upper registry shows the probability of the success of detection (the appearance of signal peak), and the lower registry shows the number of Shigella CFU nominally immobilized on the electrode. CFU values with 0.5 are mathematically calculated.



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Captions Figure 1 A cross-sectional schematic of the detection system. The Shigella cells are not drawn to scale. A metallic wire covered with an insulator is used as the gating electrode.

Figure 2 The screen-printed electrode (SPE). The sandwich immune complex is formed on the working electrode.

Figure 3 The effect of VG (= 0.6 V) on the detection signal. (a) CVs of a SPE immobilized with 3x104 CFU/mL of Shigella in PBS. (b) CVs of a SPE immobilized with 100 CFU/mL of Shigella in PBS.

Figure 4

(a) Calibration curve of Shigella in synthesized stool. (b) Stability of the detecting

electrodes tested over a period of 21 days. Three detecting SPEs were tested with 3x104 CFU/mL of Shigella. The CVs were obtained with VG =0.6V.

Figure 5 (a) Calibration curve of Shigella in human blood. The curve covers 20-3x104 CFU/mL. (b) Calibration curve in the low concentration range.


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Bacteria

HRP detection antibody

Capture antibody

HRP

Figure 1



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1 2

(a)

1 2 3

(b)

Figure 3



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Figure 4


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