Cheng Cheng, Ph.D.

RESEARCH

A highly sensitive aptasensor for on‐site detection of lipopolysaccharides in food

A rapid, low‐cost, highly sensitive, and specific capacitive aptasensor is presented for detection of lipopolysaccharides (LPS). Exposure to LPS could cause fever, gram‐negative sepsis, septic shock, and eventual death. Hence, rapid, low cost, and sensitive detection of LPS is pivotal for the safety of food, pharmaceutical, and medical devices and products. In this work, a capacitive sensing method based on alternating current electrokinetics is developed to achieve rapid and specific detection of LPS. This method uses an alternating current signal for two purposes. One is to induce positive dielectrophoresis, which attracts LPS toward the sensor electrodes’ surface and accelerates its binding with the immobilized aptamer probe. The other purpose is to simultaneously sense the binding reaction by measuring the interfacial capacitance change on the electrodes’ surface. The testing procedures and instrumentation setup of this sensing platform are significantly simplified while finding quantitative concentrations of both analytical and complex samples within 30 s. When testing analytical samples of LPS from Escherichia coli O55:B5, a LOD of 4.93 fg/mL is achieved. The recovery analysis is also performed with LPS spiked in a complex matrix and good recovery rates are demonstrated. This work provides an affordable and field‐deployable platform for highly sensitive and real‐time LPS detection.

Figure 1. The procedure of preparing and collecting LPS from a complex matrix surface. First, 0, 2.0 and 4.0 μL of 100 pg/mL LPS in 0.1 × PBS were added on one side of each spinach leaf in separate weighing boats. Samples were then covered by sarin wrap and kept into a refrigerator. Secondly, after being stored in the refrigerator at 4°C for 12 h, leaves were removed from the weighing boats and the area where LPS was originally added was cut and transferred into a 2.0 mL Eppendorf tube by a pair of forceps. Thirdly, each Eppendorf tube was filled with 500 μL 0.1 × PBS, with the spot where LPS was added completely immersed, and then spun at 2000 rpm in a desktop centrifuge for 10 min. Lastly, the supernatant was transferred into new clean tubes to be tested. The corresponding concentrations of LPS spiked on the surface of spinach leaves would be 420 and 840 fg/mL respectively.

Figure 2. (A) Dose response of LPS in 0.1 × PBS. The LOD is 4.93 fg/mL corresponding to a cut-off response of −0.027%/min. Dummy sensors tested with 1 pg/mL LPS yielded a response of 0.51 ± 0.27%/min, which is below the cut-off line. (B) Tests of spiked and unspiked LPS collected from spinach leaves (inset: Interpreted concentrations and recovery tests).

Capacitive DNA sensor for rapid and sensitive detection of whole genome human herpesvirus‐1 dsDNA in serum

This work presents a rapid, highly sensitive, low‐cost, and specific capacitive DNA sensor for detection of whole genome human herpesvirus‐1 DNA. This sensor is capable of direct DNA detection with a response time of 30 s, and it can be used to test standard buffer or serum samples. The sensing approach for DNA detection is based on alternating current (AC) electrokinetics. By applying an inhomogeneous AC electric field on sensor electrodes, positive dielectrophoresis is induced to accelerate DNA hybridization. The same applied AC signal also directly measures the hybridization of target with the probe on the sensor surface. Experiments are conducted to optimize the AC signal, as well as the buffers for probe immobilization and target DNA hybridization. The assay is highly sensitive and specific, with no response to human herpesvirus‐2 DNA at 5 ng/mL and a LOD of 1.0 pg/mL (6.5 copies/μL or 10.7 aM) in standard buffer. When testing the double stranded (ds) DNA spiked in human serum samples, the sensor yields a LOD of 20.0 pg/mL (129.5 copies/μL or 0.21 femtomolar (fM)) in neat serum. In this work, the target is whole genome dsDNA, consequently the test can be performed without the use of enzyme or amplification, which considerably simplifies the sensor operation and is highly suitable for point of care disease diagnosis.

Figure (A) Evaluation of sensor’s performances when probe is prepared in 0.05 × PBS and DNA samples in 0.5 × SSC (black), probe in 0.05 × PBS and DNA samples in 2 × SSC (red), probe in ultrapure water and DNA samples in 0.5 × SSC (blue), and probe in ultrapure water and DNA samples in 1 × SSC (green). Probe prepared in 0.05 × PBS and DNA samples in 0.5 × SSC (black) is the optimized with LOD of 1 pg/mL (6.47 copies/μL or 10.7 aM). (B) Dose response of HSV-1 DNA serum samples with 1:20 and 1:10 dilution with LOD of 20 pg/mL (129.47 copies/μL or 0.214 fM) in serum with 1:20 dilution. DNA concentration shown on x-axis refers to the original DNA concentration in neat serum.

A PCR-free point-of-care capacitive immunoassay for influenza A virus

This article describes a highly sensitive and specific capacitive immunosensor for rapid, low cost and simple-to-use detection of virus particles from clinical swab samples. An inhomogeneous AC electric field is applied on sensor electrodes. This induces positive dielectrophoresis that attracts virus particles to the sensor electrodes. As a result, rapid and sensitive detection of influenza A virus is accomplished without the need for nucleotide isolation and amplification. The same AC signal is used to detect the binding of virus particle to the sensor surface immobilized with the antibody probe. The assay is highly suitable for point-of-care use. When testing clinical swab samples, the response of samples at various dilutions is analyzed, and an optimal dilution is found and used for subsequent blind tests of clinical swab samples. Analytical experiments on standard influenza virus sample demonstrate a limit of detection of 0.25 pg/mL. Other figures of merit include (a) an assay time of 30 seconds; (b) a diagnostic sensitivity of 90%; and (c) a specificity of 70%. Blind tests are conducted for a panel of twenty nasal swab samples, and the results are in good agreement with those by using the commercial reverse transcription polymerase chain reaction.

Figure (A) Capacitive sensing mechanism and equivalent circuit of the commercially available surface acoustic wave (SAW) electrode chip. (B) Dose response of influenza A samples as a function of concentration with an LOD of 0.2513 pg/mL. (C) & (D) Comparison of results from ACEK capacitive sensors and those from commercial tests for a blind panel test of influenza virus A from nasal swabs. (C) Responses of all tested samples differentiated by the −0.40%/min cut-off line (blue) and (D) correlation between PCR cycles and responses of samples determined as positive by ACEK capacitive sensor in blind tests. The strongest positive sample is the limit of a commercial rapid influenza test.

Unamplified RNA Sensor for On‐Site Screening of Zika Virus Disease in a Limited Resource Setting

Recent outbreaks of Zika virus have been declared a public health emergency of international concern. The diagnosis of Zika infection is based on a person’s recent travel history, symptoms, and laboratory test results. However, the diagnosis of Zika infection may be delayed because symptoms are often mild and nondescript, and confirmatory laboratory tests are relatively time-consuming and expensive. Given the lack of an effective vaccine against Zika virus, and a relatively short period of viremia, developing a rapid and sensitive means of detecting the Zika virus in serum is a public health priority. This work presents a novel RNA sensor, based on a sequence-specific probe and AC electrokinetics-enhanced capacitive sensing technology to directly capture and detect Zika virus RNA. This method allows detection and quantification of Zika virus RNA in only 30 seconds, with a low limit of detection (LOD) reaching 158.1 copies/μL. The sensor is also tested for its specificity, showing no false-positive signals from other viruses. In addition, the biosensor is portable, inexpensive, and simple to use, without the need of signal amplification, which makes it ideal for field applications.

Figure (A) Illustration of the increase in Cint due to the interfacial area change prior to surface functionalization (A0), before (Ab0), and after the binding reaction (Ab). (B) Equivalent circuit of the sensor’s electrodes. (C) Fitting (dotted line) of the equivalent circuit’s measured (solid line) impedance spectra of the electrodes cell. (D) Responses of non-specific nuclei acid (HSV-1 and dengue) and virus (influenza A), and dose-response of Zika virus RNA spiked in serum/lysing solution.

Bisphenol a sensors on polyimide fabricated by laser direct writing for onsite river water monitoring at attomolar concentration

This work presents an aptamer-based, highly sensitive and specific sensor for atto- to femtomolar level detection of bisphenol A (BPA). Because of its widespread use in numerous products, BPA enters surface water from effluent discharges during its manufacture, use, and from waste landfill sites throughout the world. On-site measurement of BPA concentrations in water is important for evaluating compliance with water quality standards or environmental risk levels of the harmful compound in the environment. The sensor in this work is porous, conducting, interdigitated electrodes that are formed by laser-induced carbonization of flexible polyimide sheets. BPA-specific aptamer is immobilized on the electrodes as the probe, and its binding with BPA at the electrode surface is detected by capacitive sensing. The binding process is aided by ac electroosmotic effect that accelerates the transport of BPA molecules to the nanoporous graphene-like structured electrodes. The sensor achieved a limit of detection of 58.28 aM with a response time of 20 s. The sensor is further applied for recovery analysis of BPA spiked in surface water. This work provides an affordable platform for highly sensitive, real time, and field-deployable BPA surveillance critical to the evaluation of the ecological impact of BPA exposure.

Figure 1. (A) Schematic illustration of the laser direct writing of the electrode on a polyimide substrate, (B) typical photo image of flexible electrodes, (C) SEM images of the porous carbonized structures with scale bars of (C) and insert image (high magnification) to be 100 and 20 μm, respectively, and (D) Raman spectra of the polyimide regimes with and without laser irradiation.

Figure 2. (A) Ultrasensitive capacitive sensors with directed movement of complex sample particles with the applied ACEO effect. (B) Normalized capacitance changes of control, BPA, BPS, and BPF samples with time. (C) Detection response of BPA samples as a function of concentration. (D) Illustration of sensor specificity. The blue line is the sensor response of LOD (58.28 aM).

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