Ariana M. Nicolini

Paper microfluidic extraction and direct smartphone-based identification of pathogenic nucleic acids from field and clinical samples

A rapid, paper microfluidic- and smartphone-based protocol was developed for the extraction and direct fluorescent identification of the nucleic acids of Salmonella Typhimurium from field and clinical samples. Initially, liquid samples (10% diluted) from fresh poultry packaging were loaded on the paper chips and were lysed with Tris–EDTA (TE) buffer. Nucleic acids from the lysed samples were eluted through the paper channel with TE buffer and the paper channel was excised into three pieces for the further polymerase chain reaction (PCR) assay. The extraction efficiency was determined by measuring fluorescence reflectance with either a benchtop optical detection system (consisting of an LED light source, a pair of optical fibers, and a miniature spectrophotometer, all built on micro-positioning stages) or a smartphone-based fluorescent microscope (in-house fabricated). The limit of detection of Salmonella Typhimurium in 10% poultry packaging liquid with cellulose paper was 103 CFU mL−1, while that extracted with nitrocellulose paper was 104 CFU mL−1 (as determined by both PCR and fluorescence reflectance). Cellulose channels proved more appropriate for measuring low and very high concentrations of pathogen DNA, while nitrocellulose proved better for analysing the mid-range concentrations. We observed that DNA migrated through nitrocellulose at a faster rate and further than through cellulose due to charge–charge repulsion between nitrocellulose and DNA (both negatively charged), thus contributing to consistent and efficient extraction. We tested the efficiency of Salmonella extraction from 10% poultry packaging liquid, 10% whole blood, and 10% fecal samples, and obtained comparable extraction efficiency, as confirmed by smartphone-based direct fluorescent detection. This protocol is suitable for the direct detection of total bacteria count in a dirty sample (when specificity is not necessary) as well as determining extraction efficiency. This protocol is compatible with PCR, to provide specific information about the type of pathogen present in sample.

Droplet-based immunoassay on a 'sticky' nanofibrous surface for multiplexed and dual detection of bacteria using smartphones.

We have developed a rapid, sensitive, and specific droplet-based immunoassay for the detection of Escherichia coli and Salmonella within a single-pipetted sample. Polycaprolactone (PCL) electrospun fibers on indium-tin-oxide (ITO) glass provide a sufficient surface to render a non-slip droplet condition, and while the PCL fibers lend a local hydrophilicity (contact angle θ=74°) for sufficient sub-micron particle adhesion, air pockets within the fibers lend an apparent hydrophobicity. Overall, the contact angle of water on this electrospun surface is 119°, and the air pockets cause the droplet to be completely immobile and resistant to movement, protecting it from external vibration. By using both anti-E. coli conjugated, 510 nm diameter green fluorescent particles (480 nm excitation and 520 nm emission) and anti-Salmonella conjugated, 400 nm diameter red fluorescent particles (640 nm excitation and 690 nm emission), we can detect multiple targets in a single droplet. Using appropriate light sources guided by fiber optics, we determined a detection limit of 10(2) CFU mL(-1). Immunoagglutination can be observed under a fluorescence microscope. Fluorescence detection (at the emission wavelength) of immunoagglutination was maximum at 90° from the incident light, while light scattering (at the excitation wavelength) was still present and behaved similarly, indicating the ability of double detection, greatly improving credibility and reproducibility of the assay. A power function (light intensity) simulation of elastic Mie scatter confirmed that both fluorescence and light scattering were present. Due to the size of the fluorescent particles relative to their incident excitation wavelengths, Mie scatter conditions were observed, and fluorescence signals show a similar trend to light scattering signals. Smartphone detection was included for true portable detection, in which the high contact angle pinning of the droplet makes this format re-usable and re-configurable.

In situ, dual-mode monitoring of organ-on-a-chip with smartphone-based fluorescence microscope

The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidneys response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc.) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ. A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses.

Future developments in biosensors for field-ready Zika virus diagnostics

Since early reports of the recent Zika virus outbreak in May 2015, much has been learned and discussed regarding Zika virus infection and transmission. However, many opportunities still remain for translating these findings into field-ready sensors and diagnostics. In this brief review, we discuss current diagnostic methods, consider the prospects of translating other flavivirus biosensors directly to Zika virus sensing, and look toward the future developments needed for high-sensitivity and high-specificity biosensors to come.

Rapid and Sensitive Detection of H1N1/2009 Virus from Aerosol Samples with a Microfluidic Immunosensor.

Influenza A H1N1/2009 is a highly infectious, rapidly spreading airborne disease that needs to be monitored in near real time, preferably in a microfluidic format. However, such demonstration is difficult to find as H1N1 concentration in aerosol samples is extremely low, with interference from dust particles. In this work, we measured Mie scatter intensities from a microfluidic device with optical waveguide channels, where the antibody-conjugated latex beads immunoagglutinated with the target H1N1 antigens. Through careful optimizations of optical parameters, we were able to maximize the Mie scatter increase from the latex immunoagglutinations while minimizing the background scatter from the dust particles. The aerosol samples were collected from a 1:10 mock classroom using a button air sampler, where a nebulizer generated aerosols, simulating human coughing. The detection limits with real aerosol samples were 1 and 10 pg/mL, using a spectrometer or a cell phone camera as an optical detector, respectively. These are several orders of magnitudes more sensitive than the other methods. The microfluidic immunosensor readings are in concordance with the results of reverse transcription polymerase chain reaction. The assay time was 30 s for sampling and 5 min for the microfluidic assay.

Accessible Web Page Design for the Visually Impaired: A Case Study

Section 508 of the Rehabilitations Act of 1973 states that federal agencies are required to maintain accessible web-based information for persons with disabilities, namely, visual impairments. Studies spanning over 1 decade conducted by The American Foundation for the Blind and Towson University’s Universal Usability Lab investigated federal home pages for Section 508 violations. Both studies concluded that numerous university, corporate, federal, and federal contractor websites are largely inaccessible to people with disabilities—specifically in terms of clarity, consistency, and fidelity to standards. Due to inconsistencies across federal agencies, constant website updates, and webmaster turnaround, there is a need for practical guidelines for web page design compliant with Section 508, the Americans with Disabilities Act, and the World Wide Web Consortium’s Web Content Accessibility Guidelines, with particular focus on the visually impaired.

Tuneable nanoparticle-nanofiber composite substrate for improved cellular adhesion

This work presents a novel technique using a reverse potential electrospinning mode for fabricating nanoparticle-embedded composites that can be tailored to represent various fiber diameters, surface morphologies, and functional groups necessary for improved cellular adhesion. Polycaprolactone (PCL) nanofibers were electrospun in both traditional positive (PP) and reverse potential (RP) electrical fields. The fibers were incorporated with 300nm polystyrene (PS) fluorescent particles, which contained carboxyl, amine groups, and surfactants. In the unconventional RP, the charged colloidal particles and surfactants were shown to have an exaggerated effect on Taylor cone morphology and fiber diameter caused by the changes in charge density and surface tension of the bulk solution. The RP mode was shown to lead to a decrease in fiber diameter from 1200±100nm (diameter±SE) for the nanofibers made with PCL alone to 440±80nm with the incorporation of colloidal particles, compared to the PP mode ranging from 530±90nm to 350±50nm, respectively. The nanoparticle-nanofiber composite substrates were cultured with human umbilical vein endothelial cells (HUVECs) and evaluated for cellular viability and adhesion for up to 5 days. Adhesion to the nanofibrous substrates was improved by 180±10% with the addition of carboxylated particles and by 480±60% with the functionalization of an RGD ligand compared to the PCL nanofibers. The novel approach of electrospinning in the RP mode with the addition of colloids in order to alter charge density and surface tension could be utilized towards many applications, one being implantable biomaterials and tissue engineered scaffolds as demonstrated in this work.

Mie Scatter and Interfacial Tension Based Real‐Time Quantification of Colloidal Emulsion Nucleic Acid Amplification

This work demonstrates for the first time rapid, real‐time Mie scatter sensing of colloidal emulsion nucleic acid amplification directly from emulsion droplets. Loop‐mediated isothermal amplification is used in this study, and, to our knowledge, has not previously been used in a colloidal emulsion platform. Interfacial tension values (γ) associated with bulk protein adsorption and denaturation at the oil–water interface exhibit characteristic changes in the absence or presence of amplification. In the presence of target and amplicon, emulsions maintain a constant 300–400 nm diameter, whereas emulsions formed with no target control show a rapid decrease in droplet diameter to <100 nm over the first 20 min of incubation. This method is validated using whole bacteria (Staphylococcus aureus MSSA and Escherichia coli O157:H7) and whole virus (Potato virus Y and Zika virus) samples suspended in water, buffer, or serum‐like matrices. Short‐term formation of colloidal emulsion is quantified via 60° scatter monitoring, where the initial slope of scattering intensity is utilized to confirm target amplification in less than 5 min. The unique benefits of this method render it more cost‐effective and field‐deployable than existing methods, while being adaptable to a multitude of targets, sample matrices, and nucleic acid amplification tests.