Tu San Park

Cell-phone-based measurement of TSH using Mie scatter optimized lateral flow assays.

Semi-quantitative thyr oid stimulating hormone (TSH) lateral flow immunochromatographic assays (LFA) are used to screen for serum TSH concentration >5 mIUL(-1) (hypothyroidism). The LFA format, however, is unable to measure TSH in the normal range or detect suppressed levels of TSH (<0.4 mIU L(-1); hyperthyroidism). In fact, it does not provide quantitative TSH values at all. Obtaining quantitative TSH results, especially in the low concentration range, has until now required the use of centralized clinical laboratories which require specimen transport, specialized equipment and personnel, and result in increased cost and delays in the timely reporting of important clinical results. We have conducted a series of experiments to develop and validate an optical system and image analysis algorithm based upon a cell phone platform. It is able to provide point-of-care quantitative TSH results with a high level of sensitivity and reproducibility comparable to that of a clinical laboratory-based third-generation TSH immunoassay. Our research approach uses the methodology of the optimized Rayleigh/Mie scatter detection by taking into consideration the optical characteristics of a nitrocellulose membrane and gold nanoparticles on an LFA for quantifying TSH levels. Using a miniature spectrometer, LED light source, and optical fibers on a rotating benchtop apparatus, the light intensity from different angles of incident light and angles of detection to the LFA were measured. The optimum angles were found that the minimized Mie scattering from nitrocellulose membrane, consequently maximizes the Rayleigh scatter detection from the gold nanoparticles in the LFA bands. Using the results from the benchtop apparatus, a cell-phone-based apparatus was designed which utilized the embedded flash in the cell phone camera as the light source, piped the light with an optical fiber from the flash through a collimating lens to illuminate the LFA. Quantification of TSH was performed in an iOS application directly on the phone and verified using the code written in MATLAB. The limit of detection of the system was determined to be 0.31 mIU L(-1) (never achieved before on an LFA format), below the commonly accepted minimum concentration of 0.4 mIU L(-1) indicating clinical significance of hyperthyroidism. The system was further evaluated using human serum showing an accurate and reproducible platform for rapid and point-of-care quantification of TSH using a cell phone.

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.

Rapid and reagentless detection of microbial contamination within meat utilizing a smartphone-based biosensor

A smartphone-utilized biosensor was developed for detecting microbial spoilage on ground beef, without using antibodies, microbeads or any other reagents, towards a preliminary screening tool for microbial contamination on meat products, and potentially towards wound infection. Escherichia coli K12 solutions (101–108 CFU/mL) were added to ground beef products to simulate microbial spoilage. An 880 nm near infrared LED was irradiated perpendicular to the surface of ground beef, and the scatter signals at various angles were evaluated utilizing the gyro sensor and the digital camera of a smartphone. The angle that maximized the Mie scatter varied by the E. coli concentration: 15° for 108 CFU/mL, 30° for 104 CFU/mL, and 45° for 10 CFU/mL, etc. SEM and fluorescence microscopy experiments revealed that the antigens and cell fragments from E. coli bonded preferably to the fat particles within meat, and the size and morphologies of such aggregates varied by the E. coli concentration.

Smartphone-based, sensitive µPAD detection of urinary tract infection and gonorrhea.

The presence of bacteria in urine can be used to monitor the onset or prognosis of urinary tract infection (UTI) and some sexually-transmitted diseases (STDs), such as gonorrhea. Typically, bacterias presence in urine is confirmed by culturing samples overnight on agar plates, followed by a microscopic examination. Additionally, the presence of Escherichia coli in a urine sample can be indirectly confirmed through assaying for nitrite (generated by reducing nitrate in urine), however this is not sufficiently specific and sensitive. Species/strains identification of bacteria in a urine sample provides insight to appropriate antibiotic treatment options. In this work, a microfluidic paper analytical device (µPAD) was designed and fabricated for evaluating UTI (E. coli) and STD (Neisseria gonorrhoeae) from human urine samples. Anti-E. coli or anti-N. gonorrhoeae antibodies were conjugated to submicron particles then pre-loaded and dried in the center of each paper microfluidic channel. Human urine samples (undiluted) spiked with E. coli or N. gonorrhoeae were incubated for 5 min with 1% Tween 80. The bacteria-spiked urine samples were then introduced to the inlet of paper microfluidic channel, which flowed through the channel by capillary force. Data confirms that proteins were not filtered by μPAD, which is essential for this assay. Urobilin, the component responsible for the yellow appearance of urine and green fluorescence emission, was filtered by μPAD, resulting in significantly minimized false-positive signals. This filtration was simultaneously made during the μPAD assay and no pretreatment/purification step was necessary. Antibody-conjugated particles were immunoagglutinated at the center of the paper channel. The extent of immunoagglutination was quantified by angle-specific Mie scatter under ambient lighting conditions, utilizing a smartphone camera as a detector. The total μPAD assay time was less than 30s. The detection limit was 10 CFU/mL for both E. coli and N. gonorrhoeae, while commercially available gonorrhea rapid kit showed a detection limit of 10(6) CFU/mL. A commercially available nitrite assay test strip also had a detection limit of 10(6) CFU/mL, but this method is not antibody-based and thus not sufficiently specific. By optimizing the particle concentration, we were also able to extend the linear range of the assay up to 10(7) CFU/mL. The proposed prototype will serve as a low-cost, point-of-care, sensitive urinalysis biosensor to monitor UTI and gonorrhea from human urine.

Smartphone detection of Escherichia coli from field water samples on paper microfluidics

Smartphone detection of Escherichia coli from field water samples is successfully demonstrated using paper microfluidics. A three-channel paper chip is designed and fabricated, with a negative control channel preloaded with bovine serum albumin (BSA)-conjugated beads and two E. coli detection channels preloaded with anti-E. coli-conjugated beads, for low- and high-concentration detection. Field water samples are introduced to the paper chip by dipping or pipetting, and the antigens from E. coli travel through the paper fibers by capillary action while the dust/soil or algae particles are effectively filtered. Antibody-conjugated beads, confined within the paper fibers, immunoagglutinate in the presence of E. coli antigens, while BSA-conjugated beads do not. The extent of immunoagglutination is quantified by evaluating Mie scatter intensity from the digital images taken at an optimized angle and distance using a smartphone. The assay results show excellent agreement with the MacConkey plate results, i.e., the count of viable E. coli. The scatter simulation procedure is introduced to substitute for experimental optimization, such that the proposed method can be easily adapted to the other types of samples. A smartphone application is developed, incorporating the internal gyroscope of a smartphone, to allow the user to position the smartphone at an optimized angle of scatter detection. The detection limit is single-cell-level and the total assay time is 90 s.

Smartphone-Based Optofluidic Lab-on- a-Chip for Detecting Pathogens from Blood

A novel smartphone-based detection device was created to detect infectious pathogens directly from diluted (10%) human whole blood. The model pathogen was histidine-rich protein 2 (HRP-2), an antigen specific to Plasmodium falciparum (malaria). Anti–HRP-2–conjugated submicrobeads were mixed with HRP-2–infused 10% blood in a lab-on-a-chip device. The white LED flash and the digital camera of the smartphone were used as light source and detector, which delivered light to and from the bead and blood mixture via optofluidic channels in the lab-on-a-chip. The optofluidic channels were angled at 45 degrees to capture the Mie scatter from the sample. Considering the absorption and scattering characteristics of blood (red/infrared preferred) and the Mie scatter simulations for microbead immunoagglutination (UV preferred), blue detection showed the best results. The detection limit was 1 pg/mL in 10% blood. The linear range was from 1 pg/mL to 10 ng/mL. A handheld device, easily attachable to a single smartphone, was finally designed and fabricated using optical mirrors and lenses and successfully detected the HRP-2 from 10% blood. The total assay time was approximately 10 min. The proposed device can potentially be used for detecting a wide range of blood infection with high sensitivity.

Paper microfluidics for red wine tasting

A paper microfluidic chip was designed and fabricated to evaluate the taste of 10 different red wines using a set of chemical dyes. The digital camera of a smartphone captured the images, and its red-green-blue (RGB) pixel intensities were analyzed by principal component analysis (PCA). Using 8 dyes and 2 principal components (PCs), we were able to distinguish each wine by the grape variety and the oxidation status. Through comparing with the flavor map by human evaluation, PC1 seemed to represent the sweetness and PC2 the bodyness of red wine. This superior performance is attributed to: (1) careful selection of commercially available dyes through a series of linear correlation studies with the taste chemicals in red wines, (2) minimization of sample-to-sample variation by splitting a single sample into multiple wells on the paper microfluidics, and (3) filtration of particulate matter through paper fibers. The image processing and PCA procedure can eventually be implemented as a stand-alone smartphone application and can be adopted as an extremely low-cost, disposable, fully handheld, easy-to-use, yet sensitive and specific quality control method for appraising red wine or similar beverage products in resource-limited environments.

Smartphone Detection of UV LED-Enhanced Particle Immunoassay on Paper Microfluidics

Use of a smartphone as an optical detector for paper microfluidic devices has recently gained substantial attention due to its simplicity, ease of use, and handheld capability. Utilization of a UV light source enhances the optical signal intensities, especially for the particle immunoagglutination assay that has typically used visible or ambient light. Such enhancement is essential for true assimilation of assays to field deployable and point-of-care applications by greatly reducing the effects by independent environmental factors. This work is the first demonstration of using a UV LED (UVA) to enhance the Mie scatter signals from the particle immunoagglutination assay on the paper microfluidic devices and subsequent smartphone detection. Smartphone’s CMOS camera can recognize the UVA scatter from the paper microfluidic channels efficiently in its green channel. For an Escherichia coli assay, the normalized signal intensities increased up to 50% from the negative signal with UV LED, compared with the 4% to 7% with ambient light. Detection limit was 10 colony-forming units/mL. Similar results were obtained in the presence of 10% human whole blood.

Multi-Normalization and Interpolation Protocol to Improve Norovirus Immunoagglutination Assay from Paper Microfluidics with Smartphone Detection:

Norovirus (NoV) is one of the leading causes of acute gastroenteritis, affecting 685 million people per year around the world. The best preventive measure is to screen water for possible NoV contamination, not from infected humans, preferably using rapid and field-deployable diagnostic methods. While enzyme immunoassays (EIAs) can be used for such detection, the low infectious dose as well as the generally inferior sensitivity and low titer of available NoV antibodies render critical challenges in using EIAs toward NoV detection. In this work, we demonstrated smartphone-based Mie scatter detection of NoV with immunoagglutinated latex particles on paper microfluidic chips. Using only three different concentrations of anti-NoV–conjugated particles, we were able to construct a single standard curve that covered seven orders of magnitude of NoV antigen concentrations. Multiple normalization steps and interpolation procedures were developed to estimate the optimum amount of antibody-conjugated particles that matched to the target NoV concentration. A very low detection limit of 10 pg/mL was achieved without using any concentration or enrichment steps. This method can also be adapted for detection of any other virus pathogens whose antibodies possess low sensitivity and low antibody titer.