SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal control.

Nucleic acid amplification tests for the detection of SARS-CoV-2 have been an important testing mechanism for the COVID-19 pandemic. While these traditional nucleic acid diagnostic methods are highly sensitive and selective, they are not suited to home or clinic-based uses. Comparatively, rapid antigen tests are cost-effective and user friendly but lack in sensitivity and specificity. Here we report on the development of a one-pot, duplexed reverse transcriptase recombinase polymerase amplification SARS-CoV-2 assay with MS2 bacteriophage as a full process control. Detection is carried out with either real-time fluorescence or lateral flow readout with an analytical sensitivity of 50 copies per reaction. Unlike previously published assays, the RNA-based MS2 bacteriophage control reports on successful operation of lysis, reverse transcription, and amplification. This SARS-CoV-2 assay features highly sensitive detection, visual readout through an LFA strip, results in less than 25 minutes, minimal instrumentation, and a useful process internal control to rule out false negative test results.

REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention.

Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in HIV care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short- and long-term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase enzyme. Here, we introduce a probabilistic model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate, emtricitabine triphosphate, lamivudine triphosphate, and azidothymidine triphosphate with agreement between experiment and theory. RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring in HIV care and could be extended to other diseases treated with nucleotide analogs.

Rapid detection of hepatitis C virus using recombinase polymerase amplification.

Over 71 million people are infected with hepatitis C virus (HCV) worldwide, and approximately 400,000 global deaths result from complications of untreated chronic HCV. Pan-genomic direct-acting antivirals (DAAs) have recently become widely available and feature high cure rates in less than 12 weeks of treatment. The rollout of DAAs is reliant on diagnostic tests for HCV RNA to identify eligible patients with viremic HCV infections. Current PCR-based HCV RNA assays are restricted to well-resourced central laboratories, and there remains a prevailing clinical need for expanded access to decentralized HCV RNA testing to provide rapid chronic HCV diagnosis and linkage to DAAs in outpatient clinics. This paper reports a rapid, highly accurate, and minimally instrumented assay for HCV RNA detection using reverse transcription recombinase polymerase amplification (RT-RPA). The assay detects all HCV genotypes with a limit of detection of 25 copies per reaction for genotype 1, the most prevalent in the United States and worldwide. The clinical sensitivity and specificity of the RT-RPA assay were both 100% when evaluated using 78 diverse clinical serum specimens. The accuracy, short runtime, and low heating demands of RT-RPA may enable implementation in a point-of-care HCV test to expand global access to effective treatment via rapid chronic HCV diagnosis.

Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and viral RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

HIV pre-exposure prophylaxis adherence test using reverse transcription isothermal amplification inhibition assay.

Current HIV antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) therapy adherence monitoring relies on either patient self-reported adherence or monitored drug dispensing, which are not reliable. We report a proof-of-concept adherence monitoring assay which directly measures nucleotide reverse transcriptase inhibitor (NRTI) concentration using a reverse transcription isothermal amplification inhibition assay. We measure the concentration of Tenofovir diphosphate (TFV-DP) - an NRTI that functions as a deoxyadenosine triphosphate (dATP) analog and long-term adherence marker for PrEP - by measuring the inhibition of the reverse transcription of an RNA template. The completion or inhibition of reverse transcription is evaluated by recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification assay commonly used for point-of-care diagnostics. We present and validate a model that predicts the amplification probability as a function of dATP and TFV-DP concentrations, nucleotide insertion sites on the RNA template, and RNA template concentration. The model can be used to rationally design and optimize the assay to operate at clinically relevant TFV-DP concentrations. We provide statistical analysis that demonstrates how the assay may be used as a qualitative or semi-quantitative tool for measuring adherence to NRTI drugs and used to support patient compliance. Due to its simple instrumentation and short runtime (<1 hour), this assay has the potential for implementation in low-complexity laboratories or point-of-care settings, which may improve access to ART and PrEP adherence monitoring.

Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3,000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

Implementation and evaluation of team science training for interdisciplinary teams in an engineering design program.

INTRODUCTION: Interdisciplinary academic teams perform better when competent in teamwork; however, there is a lack of best practices of how to introduce and facilitate the development of effective learning and functioning within these teams in academic environments. METHODS: To close this gap, we tailored, implemented, and evaluated team science training in the year-long Engineering Innovation in Health (EIH) program at the University of Washington (UW), a project-based course in which engineering students across several disciplines partner with health professionals to develop technical solutions to clinical and translational health challenges. EIH faculty from the UW College of Engineering and the Institute of Translational Health Sciences' (ITHS) Team Science Core codeveloped and delivered team science training sessions and evaluated their impact with biannual surveys. A student cohort was surveyed prior to the implementation of the team science trainings, which served as a baseline. RESULTS: Survey responses were compared within and between both cohorts (approximately 55 students each Fall Quarter and 30 students each Spring Quarter). Statistically significant improvements in measures of self-efficacy and interpersonal team climate (i.e., psychological safety) were observed within and between teams. CONCLUSIONS: Tailored team science training provided to student-professional teams resulted in measurable improvements in self-efficacy and interpersonal climate both of which are crucial for teamwork and intellectual risk taking. Future research is needed to determine long-term impacts of course participation on individual and team outcomes (e.g., patents, start-ups). Additionally, adaptability of this model to clinical and translational research teams in alternate formats and settings should be tested.

Source Apportionment of Environmental Combustion Sources using Excitation Emission Matrix Fluorescence Spectroscopy and Machine Learning.

The link between particulate matter (PM) air pollution and negative health effects is well-established. Air pollution was estimated to cause 4.9 million deaths in 2017 and PM was responsible for 94% of these deaths. In order to inform effective mitigation strategies in the future, further study of PM and its health effects is important. Here, we present a method for identifying sources of combustion generated PM using excitation-emission matrix (EEM) fluorescence spectroscopy and machine learning (ML) algorithms. PM samples were collected during a health effects exposure assessment panel study in Seattle. We use archived field samples from the exposure study and the associated positive matrix factorization (PMF) source apportionment based on X-ray fluorescence and light absorbing carbon measurements to train convolutional neural network and principal component regression algorithms. We show EEM spectra from cyclohexane extracts of the archived filter samples can be used to accurately apportion mobile and vegetative burning sources but were unable to detect crustal dust, Cl-rich, secondary sulfate and fuel oil sources. The use of this EEM-ML approach may be used to conduct PM exposure studies that include source apportionment of combustion sources.

HIV detection from human serum with paper-based isotachophoretic RNA extraction and reverse transcription recombinase polymerase amplification.

The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy (ART). These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries (LMICs) has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.

Current state of commercial point-of-care nucleic acid tests for infectious diseases.

The COVID-19 pandemic has put the spotlight on the urgent need for integrated nucleic acid tests (NATs) for infectious diseases, especially those that can be used near patient ("point-of-care", POC), with rapid results and low cost, but without sacrificing sensitivity or specificity of gold standard PCR tests. In the US, the Clinical Laboratory Improvement Amendments Certificate of Waiver (CLIA-waiver) is mandated by the Food and Drug Administration (FDA) and designated to any laboratory testing with high simplicity and low risk for error, suitable for application in the POC. Since the first issuance of CLIA-waiver to Abbot's ID NOW Influenza A&B in 2015, many more NAT systems have been developed, received the CLIA-waiver in the US or World Health Organization (WHO)'s pre-qualification, and deployed to the front line of infectious disease detection. This review highlights the regulatory process for FDA and WHO in evaluating these NATs and the technology innovation of existing CLIA-waived systems. Understanding the technical advancement and challenges, unmet needs, and the trends of commercialization facilitated through the regulatory processes will help pave the foundation for future development and technology transfer from research to the market place.

Pilot evaluation of an enzymatic assay for rapid measurement of antiretroviral drug concentrations.

OBJECTIVE: Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations-a metabolite that indicates long-term PrEP adherence. SETTING: The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle. METHODS: We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader-stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using ≥ 700 fmol/punch TFV-DP as a threshold for adequate adherence (≥ 4 doses/week). RESULTS: Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥ 700 fmol/punch by LC-MS/MS. RESTRICT fluorescence correlated with LC-MS/MS measurements (r = - 0.845, p < 0.0001). Median fluorescence was 93.3 (95% confidence interval [CI] 90.9 to 114) for samples < 700 fmol/punch and 54.4 (CI 38.0 to 72.0) for samples ≥ 700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity. CONCLUSIONS: This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

Chemokinesis-driven accumulation of active colloids in low-mobility regions of fuel gradients.

Many motile cells exhibit migratory behaviors, such as chemotaxis (motion up or down a chemical gradient) or chemokinesis (dependence of speed on chemical concentration), which enable them to carry out vital functions including immune response, egg fertilization, and predator evasion. These have inspired researchers to develop self-propelled colloidal analogues to biological microswimmers, known as active colloids, that perform similar feats. Here, we study the behavior of half-platinum half-gold (Pt/Au) self-propelled rods in antiparallel gradients of hydrogen peroxide fuel and salt, which tend to increase and decrease the rods' speed, respectively. Brownian Dynamics simulations, a Fokker-Planck theoretical model, and experiments demonstrate that, at steady state, the rods accumulate in low-speed (salt-rich, peroxide-poor) regions not because of chemotaxis, but because of chemokinesis. Chemokinesis is distinct from chemotaxis in that no directional sensing or reorientation capabilities are required. The agreement between simulations, model, and experiments bolsters the role of chemokinesis in this system. This work suggests a novel strategy of exploiting chemokinesis to effect accumulation of motile colloids in desired areas.

Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis.

Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 103 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

Excitation-Emission Matrix Spectroscopy for Analysis of Chemical Composition of Combustion Generated Particulate Matter.

Analysis of particulate matter (PM) is important for the assessment of human exposures to potentially harmful agents, notably combustion-generated PM. Specifically, polycyclic aromatic hydrocarbons (PAHs) found in ultrafine PM have been linked to cardiovascular diseases and carcinogenic and mutagenic effects. In this study, we quantify the presence and concentrations of PAHs with lower molecular weight (LMW, 126 < MW < 202) and higher molecular weight (HMW, 226 < MW < 302), i.e., smaller and larger than Pyrene, in combustion-generated PM using excitation-emission matrix (EEM) fluorescence spectroscopy. Laboratory combustion PM samples were generated in a laminar diffusion inverted gravity flame reactor (IGFR) operated on ethylene and ethane. Fuel dilution by Ar in 0% to 90% range controlled the flame temperature. The colder flames result in lower PM yields however, the PM PAH content increases significantly. Temperature thresholds for PM transition from low to high organic carbon content were characterized based on the maximum flame temperature (Tmax,c ∼ 1791 to 1857 K) and the highest soot luminosity region temperature (T*c ∼ 1600 to 1650K). Principal component regression (PCR) analysis of the EEM spectra of IGFR samples correlates to GCMS data with R2 = 0.988 for LMW and 0.998 for HMW PAHs. PCR-EEM analysis trained on the IGFR samples was applied to PM samples from woodsmoke and diesel exhaust, the model accurately predicts HMW PAH concentrations with R2 = 0.976 and overestimates LMW PAHs.

Enzymatic and Chemical-Based Methods to Inactivate Endogenous Blood Ribonucleases for Nucleic Acid Diagnostics.

There are ongoing research efforts into simple and low-cost point-of-care nucleic acid amplification tests (NATs) addressing widespread diagnostic needs in resource-limited clinical settings. Nucleic acid testing for RNA targets in blood specimens typically requires sample preparation that inactivates robust blood ribonucleases (RNases) that can rapidly degrade exogenous RNA. Most NATs rely on decades-old methods that lyse pathogens and inactivate RNases with high concentrations of guanidinium salts. Herein, we investigate alternatives to standard guanidinium-based methods for RNase inactivation using an activity assay with an RNA substrate that fluoresces when cleaved. The effects of proteinase K, nonionic surfactants, SDS, dithiothreitol, and other additives on RNase activity in human serum are reported. Although proteinase K has been widely used in protocols for nuclease inactivation, it was found that high concentrations of proteinase K are unable to eliminate RNase activity in serum, unless used in concert with denaturing concentrations of SDS. It was observed that SDS must be combined with proteinase K, dithiothreitol, or both for irreversible and complete RNase inactivation in serum. This work provides an alternative chemistry for inactivating endogenous RNases for use in simple, low-cost point-of-care NATs for blood-borne pathogens.

Excitation Emission Matrix Fluorescence Spectroscopy for Combustion Generated Particulate Matter Source Identification.

The inhalation of particulate matter (PM) is a significant health risk associated with reduced life expectancy due to increased cardio-pulmonary disease and exacerbation of respiratory diseases such as asthma and pneumonia. PM originates from natural and anthropogenic sources including combustion engines, cigarettes, agricultural burning, and forest fires. Identifying the source of PM can inform effective mitigation strategies and policies, but this is difficult to do using current techniques. Here we present a method for identifying PM source using excitation emission matrix (EEM) fluorescence spectroscopy and a machine learning algorithm. We collected combustion generated PM2.5 from wood burning, diesel exhaust, and cigarettes using filters. Filters were weighted to determine mass concentration followed by extraction into cyclohexane and analysis by EEM fluorescence spectroscopy. Spectra obtained from each source served as training data for a convolutional neural network (CNN) used for source identification in mixed samples. This method can predict the presence or absence of the three laboratory sources with an overall accuracy of 89% when the threshold for classifying a source as present is 1.1 µg/m3 in air over a 24-hour sampling time. The limit of detection for cigarette, diesel and wood are 0.7, 2.6, 0.9 µg/m3, respectively, in air assuming a 24-hour sampling time at an air sampling rate of 1.8 liters per minute. We applied the CNN algorithm developed using the laboratory training data to a small set of field samples and found the algorithm was effective in some cases but would require a training data set containing more samples to be more broadly applicable.

Enzymatic Assay for Rapid Measurement of Antiretroviral Drug Levels.

Poor adherence to pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) can lead to human immunodeficiency virus (HIV) acquisition and emergence of drug-resistant infections, respectively. Measurement of antiviral drug levels provides objective adherence information that may help prevent adverse health outcomes. Gold-standard drug-level measurement by liquid chromatography/mass spectrometry is centralized, heavily instrumented, and expensive and is thus unsuitable and unavailable for routine use in clinical settings. We developed the REverSe TRanscrIptase Chain Termination (RESTRICT) assay as a rapid and accessible measurement of drug levels indicative of long-term adherence to PrEP and ART. The assay uses designer single-stranded DNA templates and intercalating fluorescent dyes to measure complementary DNA (cDNA) formation by reverse transcriptase in the presence of nucleotide reverse transcriptase inhibitor drugs. We optimized the RESTRICT assay using aqueous solutions of tenofovir diphosphate (TFV-DP), a metabolite that indicates long-term adherence to ART and PrEP, at concentrations over 2 orders of magnitude above and below the clinically relevant range. We used dilution in water as a simple sample preparation strategy to detect TFV-DP spiked into whole blood and accurately distinguished TFV-DP drug levels corresponding to low and high PrEP adherences. The RESTRICT assay is a fast and accessible test that could be useful for patients and clinicians to measure and improve ART and PrEP adherence.

Point-of-Care HIV Viral Load Testing: an Essential Tool for a Sustainable Global HIV/AIDS Response.

The global public health community has set ambitious treatment targets to end the HIV/AIDS pandemic. With the notable absence of a cure, the goal of HIV treatment is to achieve sustained suppression of an HIV viral load, which allows for immunological recovery and reduces the risk of onward HIV transmission. Monitoring HIV viral load in people living with HIV is therefore central to maintaining effective individual antiretroviral therapy as well as monitoring progress toward achieving population targets for viral suppression. The capacity for laboratory-based HIV viral load testing has increased rapidly in low- and middle-income countries, but implementation of universal viral load monitoring is still hindered by several barriers and delays. New devices for point-of-care HIV viral load testing may be used near patients to improve HIV management by reducing the turnaround time for clinical test results. The implementation of near-patient testing using these new and emerging technologies may be an essential tool for ensuring a sustainable response that will ultimately enable an end to the HIV/AIDS pandemic. In this report, we review the current and emerging technology, the evidence for decentralized viral load monitoring by non-laboratory health care workers, and the additional considerations for expanding point-of-care HIV viral load testing.

Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification.

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings.

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations.

Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.