A Low-Cost Micro-Volume Nephelometric System for Quantitative Immunoagglutination Assays.

Immunoassays have been widely used in scientific research and clinical diagnosis due to their versatile detection capability and high specificity. Immunoagglutination assays are kinds of immunoassay, which can simply and rapidly measure the concentration of analytes. In this work, we developed a low-cost micro-volume nephelometric system for quantitative immunoagglutination assays. We used off-the-shelf components to build the system, and the total cost of key components is only about 20 US dollars. The total detection volume in our system was as low as 3 µL, which could significantly reduce the reagent cost and required sample volume. We further evaluated the system performance via the immunoagglutination assay to measure the concentration of C-reactive protein, a plasma protein with levels rising in response to inflammation. The results demonstrated that our system could measure the concentration of analytes with relatively high sensitivity and precision within four minutes, and has high potential to be applied for clinical diagnostic tests.

Diagnosis of toxoplasmosis in pregnancy. Evaluation of latex-protein complexes by immnunoagglutination.

The aim of this work was to obtain a reagent based on latex particles for ruling out acute toxoplasmosis in pregnant women by immunoagglutination (IA). Latex-protein complexes (LPC) were previously synthesized coupling the recombinant protein of Toxoplasma gondii P22Ag and the homogenate of the parasite to latex particles with different size, chemical functionality and charge density. LPC were tested in IA assays against a panel of 72 pregnant women serum samples. Results were analysed through receiver operating characteristic curves, determining area under the curve (AUC), sensitivity, specificity positive and negative predictive values (PPV and NPV, respectively). It was observed that the antigenicity of proteins was not affected during sensitization by either physical adsorption or covalent coupling. The best results in the sense of maximizing discrimination of low avidity sera from chronic ones were observed for the IA test based on latex particles with carboxyl functionality and the recombinant P22Ag, obtaining an AUC of 0·94, a sensitivity of 100% and a NPV of 100%. In this way, the proposed test could be useful for the toxoplasmosis diagnosis in pregnant women, with the advantages of being cheap, rapid and easy to be implemented.

Development of a simple and economical diagnostic test for canine leishmaniasis.

Visceral leishmaniasis is a public health problem worldwide. The early diagnosis in dogs is crucial, since they are an epidemiologically relevant reservoir of the disease. The aim of a field study is to early identify the disease allowing rapid intervention to reduce its effects. We propose an immunoagglutination test as a visual in situ method for diagnosis of canine visceral leishmaniasis. Latex-protein complexes were sensitized by covalent coupling of a chimeric recombinant antigen of Leishmania spp. onto polystyrene latex with carboxyl functionality. The reaction time and the antigen concentration under which the immunoagglutination assay shows greater discrimination between the responses of a positive control serum and a negative control serum were determined. Then, the latex-protein complexes were evaluated as a visual diagnostic tool with a panel of 170 sera. The test may be read between 2 and 5 min and can be performed even using sera with elevated concentration of lipids, bilirubin or with variable percentage of hemolysis. The sensitivity, the specificity and the diagnostic accuracy were 78%; 100% and >80%, respectively. The visual immunoagglutination test is of potential application as a method for field studies because it shows results in less than 5 min, it is easy to implement and does not require sophisticated equipment.

Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex-(recombinant antigen) complexes.

OBJECTIVE: To determine the conditions under which the immunoagglutination assay to detect Chagas disease, obtained from a novel latex-(chimeric recombinant antigen) complex, shows greater discrimination between the responses of a positive control serum and a negative control serum. METHODS: The following variables were determined: (i) the sensitisation mechanism, (ii) the emulsifier employed for protein desorption, (iii) the reaction time, (iv) the ionic strength of the reaction medium, (v) the particle concentration, (vi) the presence of blocking agents, (vii) the presence of polyethyleneglycol as potentiator of reaction and (viii) the antigen and antibody concentrations. The search of optimal conditions was investigated by varying one variable at a time. To this effect, monodisperse latex particles sensitised with a recombinant chimeric protein (CP1) were subjected to different conditions. The agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. RESULTS: The maximum discrimination between negative and positive sera was obtained at a reaction time of 5 min, when latex complexes with a concentration of covalently coupled protein of 2.90 mg/m(2) were put in contact with undiluted sera in buffer borate pH 8-20 mm containing glycine (0.1 m) and polyethyleneglycol 8000 (3% w/v). Finally, the latex-protein complex was tested under the obtained optimal conditions, with a panel of Trypanosoma cruzi-positive sera, leishmaniasis-positive sera and -negative sera for both parasites. CONCLUSION: The immunoagglutination test based on the latex-CP1 complex could be used as a screening method for detecting Chagas disease. This test is rapid, easy to implement and could be used under field conditions; but its results should be confirmed by reference techniques like ELISA, HAI, and IFI.

Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip.

Respiratory viruses, especially coronaviruses, have resulted in worldwide pandemics in the past couple of decades. Saliva-based paper microfluidic assays represent an opportunity for noninvasive and rapid screening, yet both the sample matrix and test method come with unique challenges. In this work, we demonstrated the rapid and sensitive detection of SARS-CoV-2 from saliva samples, which could be simpler and more comfortable for patients than existing methods. Furthermore, we systematically investigated the components of saliva samples that affected assay performance. Using only a smartphone, an antibody-conjugated particle suspension, and a paper microfluidic chip, we made the assay user-friendly with minimal processing. Unlike the previously established flow rate assays that depended solely on the flow rate or distance, this unique assay analyzes the flow profile to determine infection status. Particle-target immunoagglutination changed the surface tension and subsequently the capillary flow velocity profile. A smartphone camera automatically measured the flow profile using a Python script, which was not affected by ambient light variations. The limit of detection (LOD) was 1 fg/µL SARS-CoV-2 from 1% saliva samples and 10 fg/µL from simulated saline gargle samples (15% saliva and 0.9% saline). This method was highly specific as demonstrated using influenza A/H1N1. The sample-to-answer assay time was <15 min, including <1-min capillary flow time. The overall accuracy was 89% with relatively clean clinical saline gargle samples. Despite some limitations with turbid clinical samples, this method presents a potential solution for rapid mass testing techniques during any infectious disease outbreak as soon as the antibodies become available.

Smartphone-Based Paper Microfluidic Immunoassay of Salmonella and E. coli.

Previous studies from our lab have created a simple procedure for single-cell count of bacteria on a paper chip platform using optical detection from a smartphone. The procedure and steps employed are outlined along with the lessons learned and details of certain steps and how the design was optimized. Smartphone optical detection is easy to use, low cost, and potentially field deployable, which can be useful for early and rapid detection of pathogens. Smartphone imaging of a paper microfluidic chip preloaded with antibody-conjugated particles provides an adaptable platform for detection of different bacterial targets. The paper microfluidic chip was fabricated with a multichannel design. Each channel was preloaded with either a negative control of bovine serum albumin (BSA) conjugated particles, anti-Salmonella Typhimurium-conjugated particles with varying amounts (to cover different ranges of assay), or anti-Escherichia coli-conjugated particles. Samples were introduced to the paper microfluidic chip using pipetting. Antigens of Salmonella Typhimurium traveled through the channel by capillary action confined within the paper fibers surrounded by the hydrophobic barrier. The paper channel was observed to act as a filter for unwanted particles and contaminants found in field samples. Serial dilutions of known concentrations of bacterial targets were also tested using this procedure to construct a standard curve prior to the assays. The antibody-conjugated particles were able to immunoagglutinate which was quantified through evaluation of Mie scatter intensity. This Mie scattering was quantified in images taken with a smartphone at an optimized angle and distance. Mie scatter simulation provided a method of optimizing the experimental setup and could translate easily to other types of target sample matrices. A smartphone application was developed to help the user position the smartphone optimally in relation to the paper microfluidic chip. The application integrated both image capturing capability and a simple image processing algorithm that calculated bacteria concentrations. The detection limit was at a single-cell level with a total assay time ranging from 90 to less than 60 s depending on the target.

New and rapid strategies for the diagnosis of bovine paratuberculosis "in situ" using latex particles.

The chemical coupling of a protoplasmatic antigen from Mycobacterium avium subsp. paratubeculosis onto core-shell carboxylated particles was investigated with the aim of producing latex-protein complexes to be used in immunoagglutination assays capable of detecting bovine paratuberculosis disease. For this purpose, sensitizations were carried out using both colored and not colored carboxylated latexes as well as the protoplasmatic antigen at pH close to its isoelectric point to favor the antigenic protein to approach the particle surface. In all cases, higher fractions of proteins were chemically-bound to carboxyl groups on the surface of the particles. The assessment of the performance of the visual immunoagglutination assays consisted of evaluating 111 sera from healthy and infected bovines with Mycobacterium avium subsp. paratuberculosis. Complexes obtained from the colored latex allowed an acceptable visual discrimination between the studied positive and negative sera. Most of the positive samples showed strong to very strong agglutination and only a few samples reacted weakly, i.e. a sensitivity of 70%. The specificity of the assay, on the other hand, was 86%. Therefore, this rapid detection technique allows an easy and inexpensive identification of animals possibly infected with paratuberculosis "in situ" in the herds.

Modernization of Control of Pathogenic Micro-Organisms in the Food-Chain Requires a Durable Role for Immunoaffinity-Based Detection Methodology-A Review.

Food microbiology is deluged by a vastly growing plethora of analytical methods. This review endeavors to color the context into which methodology has to fit and underlines the importance of sampling and sample treatment. The context is that the highest risk of food contamination is through the animal and human fecal route with a majority of foodborne infections originating from sources in mass and domestic kitchens at the end of the food-chain. Containment requires easy-to-use, failsafe, single-use tests giving an overall risk score in situ. Conversely, progressive food-safety systems are relying increasingly on early assessment of batches and groups involving risk-based sampling, monitoring environment and herd/flock health status, and (historic) food-chain information. Accordingly, responsible field laboratories prefer specificity, multi-analyte, and high-throughput procedures. Under certain etiological and epidemiological circumstances, indirect antigen immunoaffinity assays outperform the diagnostic sensitivity and diagnostic specificity of e.g., nucleic acid sequence-based assays. The current bulk of testing involves therefore ante- and post-mortem probing of humoral response to several pathogens. In this review, the inclusion of immunoglobulins against additional invasive micro-organisms indicating the level of hygiene and ergo public health risks in tests is advocated. Immunomagnetic separation, immunochromatography, immunosensor, microsphere array, lab-on-a-chip/disc platforms increasingly in combination with nanotechnologies, are discussed. The heuristic development of portable and ambulant microfluidic devices is intriguing and promising. Tant pis, many new platforms seem unattainable as the industry standard. Comparability of results with those of reference methods hinders the implementation of new technologies. Whatever the scientific and technological excellence and incentives, the decision-maker determines this implementation after weighing mainly costs and business risks.

Smartphone based on-chip fluorescence imaging and capillary flow velocity measurement for detecting ROR1+ cancer cells from buffy coat blood samples on dual-layer paper microfluidic chip.

Diagnosis of hematological cancer requires complete white blood cell count, followed by flow cytometry with multiple markers, and cytology. It requires substantial time and specialized training. A dual-layer paper microfluidic chip was developed as a quicker, low-cost, and field-deployable alternative to detect ROR1+ (receptor tyrosine-like orphan receptor one) cancer cells from the undiluted and untreated buffy coat blood samples. The first capture layer consisted of a GF/D glass fiber substrate, preloaded with cancer specific anti-ROR1 conjugated fluorescent particles to its center for cancer cell capture and direct smartphone fluorescence imaging. The second flow layer was comprised of a grade 1 cellulose chromatography paper with wax-printed four channels for wicking and capillary flow-based detection. The flow velocity was used as measure of antigen concentration in the buffy coat sample. In this manner, intact cells and their antigens were separated and independently analyzed by both imaging and flow velocity analyses. A custom-made smartphone-based fluorescence microscope and automated image processing and particle counter software were developed to enumerate particles on paper, with the limit of detection of 1 cell/µL. Flow velocity analysis showed even greater sensitivity, with the limit of detection of 0.1 cells/µL in the first 6 s of assay. Comparison with capillary flow model revealed great alignment with experimental data and greater correlation to viscosity than interfacial tension. Our proposed device is able to capture and on-chip image ROR1+ cancer cells within a complex sample matrix (buffy coat) while simultaneously quantifying cell concentration in a point-of-care manner.

µPAD Fluorescence Scattering Immunoagglutination Assay for Cancer Biomarkers from Blood and Serum.

A microfluidic paper analytical device (µPAD) was created for the sensitive quantification of cancer antigens, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), from human whole blood and serum, toward diagnosis and prognosis of colorectal cancer. Anti-CEA and anti-CA 19-9 antibodies were covalently linked to submicron, fluorescent polystyrene particles, loaded, and then dried in the center of the µPAD channel. CEA- or CA 19-9-spiked blood or serum samples were loaded to the inlet of µPAD, and subsequent immunoagglutination changed the fluorescent scatter signals upon ultraviolet (UV) excitation. The total assay time was about 1 min. Detection limits were 1 pg/mL for CEA and 0.1 U/mL for CA 19-9 from both 10% diluted blood and undiluted serum. The use of UV excitation and subsequent fluorescence scattering enabled much higher double-normalized intensities (up to 1.28-3.51, compared with 1.067 with the elastic Mie scatter detection), successful detection in the presence of blood or serum, and distinct multiplex assays with minimum cross-reaction of antibodies. The results with undiluted serum showed the larger dynamic range and smaller standard errors, which can be attributed to the presence of serum proteins, functioning as a stabilizer or a passivating protein for the particles within paper fibers.

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 kidney's 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.

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.

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.

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.