Projects

Aptamer-based Fluorometric Determination of Norovirus Using a Paper-based Microfluidic Device

Xuan Weng & Suresh Neethirajan

Microchimica Acta DOI: 10.1007/s00604-017-2467-x  (Link)

 

We describe a rapid and highly sensitive biosensor towards point-of-care device for rapid determination of noroviruses, a leading cause of outbreak of acute gastroenteritis worldwide. It is based on the use of a norovirus-specific aptamer labeled with 6-carboxyfluorescein, and of multi-walled carbon nanotubes (MWCNT) and graphene oxide (GO). The fluorescence of the 6-FAM labeled aptamer is quenched by MWCNT or GO via fluorescence resonance energy transfer (FRET). In the presence of norovirus, fluorescence is recovered due to the release of the labeled aptamer from MWCNT or GO. An easy-to-make paper-based microfluidic platform was developed using a nitrocellulose membrane. The quantitation of norovirus was successfully performed. The linear range extends from 12.9 ng⋅mL⁻¹ ~12.9 µg⋅mL⁻¹ of norovirus. The detection limits are 4.4 ng⋅mL⁻¹ and 3.3 ng⋅mL⁻¹, respectively, when using MWCNT or GO. The device is simple and cost-effective, and holds the potential of rapid in-situ visual determination of noroviruses with remarkable sensitivity and specificity, which provides a new method for early identification of norovirus and thereby an early intervention for preventing the spread of an outbreak.

Keywords: Biosensor; Aptamer; Norovirus; Paper-based microfluidic device; Nitrocellulose membrane; Multi-walled carbon nanotubes; Graphene oxide

 

 

 

Microfluidic platform integrated with graphene-gold nano-composite aptasensor for one-step detection of norovirus  (Link

 

Noroviruses are a foremost cause of gastroenteritis outbreaks throughout the world. On-site sample processing and detection of the viral clinical samples has always been a problem. This study reports an all-polydimethylsiloxane microfluidic chip integrated with screen-printed carbon electrode for the electrochemical detection of norovirus. The microfluidic chip contained packed silica microbeads zones to filter and enrich the norovirus infected clinical sample. Selective detection of norovirus was accomplished by functionalizing the graphene-gold nanoparticles composite modified carbon electrode with the viral capsid-specific aptamer. Norovirus specific aptamer was tagged with a ferrocene molecule, which acts a redox probe. The interaction of aptamer and norovirus resulted in a decrease in the electrochemical signal from ferrocene. The microfluidic chip and functionalized electrodes were characterized using several microscopic and electrochemical techniques. The optimized microfluidic aptasensor was employed to detect a range of norovirus concentration. Using differential pulse voltammetric analysis, a detection limit of 100  pM with a detection range from 100  pM to 3.5 nM for norovirus was obtained. The application of aptasensor was also assessed by detecting norovirus in spiked blood samples. The aptasensor could easily discriminate between the target norovirus and other interfering molecules. The developed microfluidic aptasensor has the potential to be used for point-of-care one-step detection of norovirus in clinical samples.

 

Electrochemical latent redox ratiometric probes for real-time tracking and quantification of endogenous hydrogen sulfide production in living cells

Biosensors and Bioelectronics - https://doi.org/10.1016/j.bios.2017.05.006

Hydrogen sulfide (H2S) was discovered as a third gasotransmitter in biological systems and recent years have seen a growing interest to understand its physiological and pathological functions. However, one major limiting factor is the lack of robust sensors to quantitatively track its production in real-time. We described a facile electrochemical assay based on latent redox probe approach for highly specific and sensitive quantification in living cells. Two chemical probes, Azido Benzyl ferrocene carbamate (ABFC) and N-alkyl Azido Benzyl ferrocene carbamate (NABFC) composed of azide trigger group were designed. H2S molecules specifically triggered the release of reporters from probes and the current response was monitored using graphene oxide film modified electrode as transducer. The detection limits are 0.32 µM (ABFC) and 0.076 µM (NABFC) which are comparable to those of current sensitive methods. The probes are successful in the determination of H2S spiked in whole human blood, fetal bovine serum, and E. coli. The continuous monitoring and quantification of endogenous H2S production in E. coli were successfully accomplished. This work lays first step stone towards real-time electrochemical quantification of endogenous H2S in living cells, thus hold great promise in the analytical aspects of H2S.

 

Amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus 

Syed Rahin Ahmed1, Juan C. Corredor2, Éva Nagy2, Suresh Neethirajan1* 

Nanotheranostics Journal 

Nanomaterial-based artificial enzymes or nanozymes exhibit superior properties such as stability, cost effectiveness and ease of preparation in comparison to conventional enzymes. However, the lower catalytic activity of nanozymes limits their sensitivity and thereby practical applications in the bioanalytical field. To overcome this drawback, herein we propose a very simple but highly sensitive, specific and low-cost dual enhanced colorimetric immunoassay for avian influenza A (H5N1) virus. 3,3´,5,5´- Tetramethylbenzidine (TMBZ) was used as a reducing agent to produce gold nanoparticles (Au NPs) with blue colored solution from a viral target-specific antibody-gold ion mixture at first step. The developed blue color from the sensing design was further amplified through catalytic activity of Au NPs in presence of TMBZ–hydrogen peroxide (H2O2) solution in second step. Hence, the developed dual enhanced colorimetric immunosensor enables the detection of avian influenza virus A (H5N1) with a limit of detection (LOD) of 1.11 pg/mL. Our results confirmed that the developed assay has superior sensitivity than the conventional ELISA method, plasmonic-based bioassay and commercial flu diagnostic kits. Proposed sensing method further showed its capability to detect real viruses, avian influenza A (H4N6) and A (H9N2) virus, in blood samples with limit of detection of 0.0269 HAU and 0.0331 HAU respectively. 

 

In situ self-assembly of gold nanoparticles on hydrophilic and hydrophobic substrates for influenza virus-sensing platform 

Syed Rahin Ahmed, Jeonghyo Kim, Van Tan Tran, , Tetsuro Suzuki, , Suresh Neethirajan, Jaebeom Lee, & Enoch Y. Park

Scientific Reports https://www.nature.com/articles/srep44495

 

Nanomaterials without chemical linkers or physical interactions that reside on a two-dimensional surface are attractive because of their electronic, optical and catalytic properties. An in situ method has been developed to fabricate gold nanoparticle (Au NP) films on different substrates, regardless of whether they are hydrophilic or hydrophobic surfaces, including glass, 96-well polystyrene plates, and polydimethylsiloxane (PDMS). A mixture of sodium formate (HCOONa) and chloroauric acid (HAuCl4) solution was used to prepare Au NP films at room temperature. An experimental study of the mechanism revealed that film formation is dependent on surface wettability and inter particle attraction. The as-fabricated Au NP films were further applied to the colorimetric detection of influenza virus. The response to the commercial target, New Caledonia/H1N1/1999 influenza virus, was linear in the range from 10 pg/ml to 10 μg/ml and limit of detection was 50.5 pg/ml. In the presence of clinically isolated influenza A virus (H3N2), the optical density of developed color was dependent on the virus concentration (10–50,000 PFU/ml). The limit of detection of this study was 24.3 PFU/ml, a limit 116 times lower than that of conventional ELISA (2824.3 PFU/ml). The sensitivity was also 500 times greater than that of commercial immunochromatography kits.

 

 

 

 

 

 

 

GryphSens: A Smartphone-Based Portable Diagnostic Reader for the Rapid Detection of Progesterone in Milk

Hyunwook Jang, Syed Rahin Ahmed and Suresh Neethirajan

Enzyme-linked immunosorbent assay (ELISA) is a popular assay technique for the detection and quantification of various biological substances due its high sensitivity and specificity. More often, it requires large and expensive laboratory instruments, which makes it difficult to conduct when the tests must be performed quickly at the point-of-care (POC). To increase portability and ease of use, we propose a portable diagnostic system based on a Raspberry Pi imaging sensor for the rapid detection of progesterone in milk samples. We designed, assembled, and tested a standalone portable diagnostic reader and validated it for progesterone detection against a standard ELISA assay using a commercial plate reader. The portable POC device yielded consistent results, regardless of differences in the cameras and flashlights between various smartphone devices. An Android application was built to provide front-end access to users, control the diagnostic reader, and display and store the progesterone measurement on the smartphone. The diagnostic reader takes images of the samples, reads the pixel values, processes the results, and presents the results on the handheld device. The proposed POC reader can perform to superior levels of performance as a plate reader, while adding the desirable qualities of portability and ease of use.  

     

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Bionanotechnology Laboratory
Suresh Neethirajan

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