Sam R. Nugen

PMMA biosensor for nucleic acids with integrated mixer and electrochemical detection.

This paper discusses the design, microfabrication and use of an electrochemical biosensor based on a polymer substrate for cost effectiveness and disposability. As model analyte, amplified hsp70 mRNA from Cryptosporidium parvum was chosen. Microfluidic channels were fabricated in poly(methyl methacrylate) (PMMA) using hot embossing with a copper master. The electrochemical transducer, an interdigitated ultramicroelectrode array (IDUA) was also realized directly on the PMMA surface. First, the unstructured PMMA surface was UV functionalized. An 8 min UV treatment resulted in a carboxylic acid density of approximately 8 nmol/cm(2) on the PMMA surface. The surface carboxylic acid groups were then conjugated to cystamine using water-soluble carbodiimide chemistry. Gold (200 nm) was then evaporated onto the thiol-functionalized surface. Using standard photolithography techniques, the IDUA containing 10 microm wide electrodes with 5 microm gaps was then formed followed by a gold etch. The PMMA surface containing the microchannel was subsequently bonded to the PMMA surface containing the IDUA using UV-assisted thermal bonding. The additional UV treatment also served to decrease the water contact angle of the surface from 62.5 degrees +/-0.7 degrees to 48.4 degrees +/-0.2 degrees thus, aiding with the capillary flow in the device. The hsp70 mRNA was isolated from C. parvum oocysts and amplified using nucleic acid sequence-based amplification (NASBA). The amplicon was detected in a sandwich hybridization assay with capture probe-coated superparamagnetic beads and reporter probe-tagged liposomes. The liposomes entrapped potassium ferro/ferrihexacyanide to enable amperometric quantification of the amplicon on the IDUA. Amplified mRNA from only 1 oocyst was detectable with this PMMA biosensor. The final detection device measured approximately 10 mm x 40 mm x 3 mm and contained two detection channels for dual analyses.

Detection of Escherichia coli in Drinking Water Using T7 Bacteriophage-Conjugated Magnetic Probe

In this study, we demonstrate a bacteriophage (phage)-based magnetic separation scheme for the rapid detection of Escherichia coli (E. coli) in drinking water. T7 phage is a lytic phage with a broad host range specificity for E. coli. Our scheme was as follows: (1) T7 bacteriophage-conjugated magnetic beads were used to capture and separate E. coli BL21 from drinking water; (2) subsequent phage-mediated lysis was used to release endemic β-galactosidase (β-gal) from the bound bacterial cells; (3) the release of β-gal was detected using chlorophenol red-β-d-galactopyranoside (CRPG), a colorimetric substrate which changes from yellow to red in the presence of β-gal. Using this strategy, we were able to detect E. coli at a concentration of 1 × 10(4) CFU·mL(-1) within 2.5 h. The specificity of the proposed magnetic probes toward E. coli was demonstrated against a background of competing bacteria. By incorporating a pre-enrichment step in Luria-Bertani (LB) broth supplemented with isopropyl β-d-thiogalactopyranoside (IPTG), we were able to detect 10 CFU·mL(-1) in drinking water after 6 h of pre-enrichment. The colorimetric change can be determined either by visual observation or with a reader, allowing for a simple, rapid quantification of E. coli in resource-limited settings.

Nanoimprinted Patterned Pillar Substrates for Surface-Enhanced Raman Scattering Applications

A pragmatic method to deposit silver nanoparticles on polydopamine-coated nanoimprinted pillars for use as surface-enhanced Raman scattering (SERS) substrates was developed. Pillar arrays consisting of poly(methyl methacrylate) (PMMA) that ranged in diameter from 300 to 500 nm were fabricated using nanoimprint lithography. The arrays had periodicities from 0.6 to 4.0 μm. A polydopamine layer was coated on the pillars in order to facilitate the reduction of silver ions to create silver nucleation sites during the electroless deposition of sliver nanoparticles. The size and density of silver nanoparticles were controlled by adjusting the growth time for the optimization of the SERS performance. The size of the surface-adhered nanoparticles ranged between 75 and 175 nm, and the average particle density was ∼30 particles per μm(2). These functionalized arrays had a high sensitivity and excellent signal reproducibility for the SERS-based detection of 4-methoxybenzoic acid. The substrates were also able to allow the SERS-based differentiation of three types of bacteriophages (λ, T3, and T7).

Colorimetric Detection of Escherichia coli Based on the Enzyme‐Induced Metallization of Gold Nanorods

A novel enzyme-induced metallization colorimetric assay is developed to monitor and measure beta-galactosidase (β-gal) activity, and is further employed for colorimetric bacteriophage (phage)-enabled detection of Escherichia coli (E. coli). This assay relies on enzymatic reaction-induced silver deposition on the surface of gold nanorods (AuNRs). In the presence of β-gal, the substrate p-aminophenyl β-d-galactopyranoside is hydrolyzed to produce p-aminophenol (PAP). Reduction of silver ions by PAP generates a silver shell on the surface of AuNRs, resulting in the blue shift of the longitudinal localized surface plasmon resonance peak and multicolor changes of the detection solution from light green to orange-red. Under optimized conditions, the detection limit for β-gal is 128 pM, which is lower than the conventional colorimetric assay. Additionally, the assay has a broader dynamic range for β-gal detection. The specificity of this assay for the detection of β-gal is demonstrated against several protein competitors. Additionally, this technique is successfully applied to detect E. coli bacteria cells in combination with bacteriophage infection. Due to the simplicity and short incubation time of this enzyme-induced metallization colorimetric method, the assay is well suited for the detection of bacteria in low-resource settings.

Highly sensitive and selective detection of nitrite ions using Fe3O4@SiO2/Au magnetic nanoparticles by surface-enhanced Raman spectroscopy.

A novel and pragmatic method was developed to detect the concentration of nitrite ions using Fe3O4@SiO2/Au magnetic nanoparticles (MNPs) by surface-enhanced Raman scattering (SERS). The as-prepared bifunctional nanocomposites can be used to simultaneously purify target molecules using external magnetic field and produce Raman fingerprint spectrum with trace level of target molecules. In acidic media, 4-aminothiophenol (4-ATP) molecules conjugated on Fe3O4@SiO2/Au MNPs were triggered by nitrite ions to form azo bonds, resulting in three new marker peaks on the SERS spectrum. Under optimized conditions, the detection limit based on the three marker peaks were 15.63, 13.69, and 17.77μM, which was much lower than the maximum NO2(-) concentration of 1.0mgL(-1) (71.4μM) allowed in drinking water as defined by U.S. Environmental Protection Agency (EPA). The specificity of this proposed method to detect nitrite ions was demonstrated using common ions as competitors. In addition, the SERS-based technique was successfully employed to detect nitrite ions in pond water, a synthetic urine solution, and pickle brine. Considering its good sensitivity and selectivity, the detection method is well suited for the detection of nitrite ions in real samples without pretreatment steps.

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.

Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids

Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target.

Electrospun water-soluble polymer nanofibers for the dehydration and storage of sensitive reagents

The ability to preserve and deliver reagents remains an obstacle for the successful deployment of self-contained diagnostic microdevices. In this study we investigated the ability of bacteriophage T7 to be encapsulated and preserved in water soluble nanofibers. The bacteriophage T7 was added to mixtures of polyvinylpyrrolidone and water and electrospun onto a grounded plate. Trehalose and magnesium salts were added to the mixtures to determine their effect on the infectivity of the bacteriophage following electrospinning and during storage. The loss of T7 infectivity was determined immediately following electrospinning and during storage using agar overlay plating and plaque counting. The results indicate that the addition of magnesium salts protects the bacteriophage during the relatively violent and high voltage electrospinning process, but is not as effective as a protectant during storage of the dried T7. Conversely, the addition of trehalose into the electrospinning mix has little effect on the electrospinning, but a more significant role as a protectant during storage.

Nanoscale optofluidic sensor arrays for Dengue virus detection

In this paper we present our work towards the development of nanoscale optofluidic sensor arrays (NOSAs) for Dengue virus detection. Our approach is based on the use of optically resonant devices whose resonant wavelength is shifted due to a local change in refractive index caused by a positive binding event between a surface bound molecule and it solution phase target. A special two stage micro-/nanofluidics architecture is used to first functionalize the devices and then to deliver the targets. Experimental results measuring the bulk sensitivity of the device are shown to agree extremely well with theory. Preliminary data showing the successful detection of one of the four Dengue virus serotypes is also presented. The primary advantage of these devices over the state of the art is that they combine the extensive property sensitivity (mass limit of detection) of nanosensor devices with the parallelism of the microarray format.

Water-Soluble Electrospun Nanofibers as a Method for On-Chip Reagent Storage

This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format. Polyvinylpyrrolidone (PVP) nanofibers were spun with incorporation of the enzyme horseradish peroxidase (HRP). Scanning electron microscopy (SEM) of the spun nanofibers was used to confirm the non-woven structure which had an average diameter of 155 ± 34 nm. The HRP containing fibers were tested for their change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP by reaction with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. After a storage study over 280 days, 40% of the activity remained. In addition to activity, the fibers were observed to solubilize in the microfluidic chamber. The chromogenic 3,3′,5,5′-tetramethylbenzidine solution reacted immediately with the fibers as they passed through a microfluidic channel. The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform.