Projects

Paper based microfluidic aptasensor for food safety

Xuan Weng and Suresh Neethirajan 

Journal of Food Safety (Link

 

Abstract

Food analysis is requiring rapid, accurate, sensitive and cost-effective methods to monitor and guarantee the safety and quality to fulfill the strict food legislation and consumer demands. In the present study, a nano-materials enhanced multipurpose paper based microfluidic aptasensor was developed as a sensing tool for accurate detection of food allergens and food toxins. Graphene oxide (GO) and specific aptamer-functionalized quantum dots (QDs) were employed as probes, the fluorescence quenching and recovering of the QDs caused by the interaction among GO, aptamer-functionalized QDs and the target protein were investigated to quantitatively analyze the target concentration. The homogenous assay was performed on the paper based microfluidic chip, which significantly decreased the sample and reagent consumption and reduced the assay time. Egg white lysozyme, ß-conglutin lupine and food toxins, okadaic acid and brevetoxin standard solutions and spiked food samples were successfully assayed by the presented aptasensor. Dual-target assay was completed within 5 min, and superior sensitivities were achieved when testing the samples with commercial ELISA kits side by side.

Practical applications

The present aptasensor provides a simple, accurate method for rapid quantitative analysis of allergens or toxins in food. This method is able to achieved rapid on-site detection of potential allergen/toxin contaminations, which is a critical necessity for individuals with food allergies and other types of food sensitivities. In addition, the present method can be easily implemented into routine analysis to help food producers and regulations secure the safety and compliance of food products.

 

Nanoscale imaging approaches to quantifying the electrical properties of pathogenic bacteria

Ryan Berthelot and Suresh Neethirajan

Biomedical Physics and Engineering Express  (Link

Biofilms are natural, resilient films formed when microorganisms adhere to a surface and form a complex three-dimensional structure that allows them to persist in a wide variety of environments. Readily forming in hospitals and on medical equipment, biofilms are frequent causes of infections and their subsequent complications. Due to the complexity of these structures, systematically studying individual bacterial cells and their interactions with their surrounding environment will provide a deeper understanding of the processes occurring within the biofilm as whole versus bulk population based methods that do not differentiate individual cells or species. Methods based on atomic force microscopy (AFM) are particularly suited to the study of individual cells, but are underutilized for the study of bacterial electrical properties. The ability of electrical currents to impair bacterial attachment is well documented, but to utilize electrical current as an effective antibacterial treatment, it is important to understand the electrical properties of bacteria. Therefore, we used AFM, Kelvin probe force microscopy, and ResiScope (module to perform conductive AFM) to measure the surface potential and conductance of Psuedomonas aeruginosa and methicillin resistance Staphylococcus aureus (MRSA) on gold and stainless steel. This is the first study to directly measure the electrical resistance of single bacterial cells using ResiScope. Our goal was to develop a framework for measuring biological molecules using conductive atomic force microscopy. We found that the average resistance for P. aeruginosa was 135±25 GΩ, while MRSA had an average of 173±16 GΩ. Using KPFM, the surface potential of MRSA shifted from -0.304 V to 0.153 V and from -0.280 V to 0.172 V for P. aeruginosa on gold versus stainless steel substrates, respectively. In an attempt to identify a potential charge carrier, peptidoglycan was also measured with the ResiScope module and shown to have a resistance of 105 GΩ. 

 

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.

 

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

 

 

 

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.

 

 

 

 

 

 

 

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Bionanotechnology Laboratory
Suresh Neethirajan

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