Chemically Amplified Multiplex Detection of SARS-CoV-2 and Influenza A and B Viruses via Paint-Programmed Lateral Flow Assays.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) continues to threaten lives by evolving into new variants with greater transmissibility. Although lateral flow assays (LFAs) are widely used to self-test for coronavirus disease 2019 (COVID-19), these tests suffer from low sensitivity leading to a high rate of false negative results. In this work, a multiplexed lateral flow assay is reported for the detection of SARS-CoV-2 and influenza A and B viruses in human saliva with a built-in chemical amplification of the colorimetric signal for enhanced sensitivity. To automate the amplification process, the paper-based device is integrated with an imprinted flow controller, which coordinates the routing of different reagents and ensures their sequential and timely delivery to run an optimal amplification reaction. Using the assay, SARS-CoV-2 and influenza A and B viruses can be detected with ≈25x higher sensitivity than commercial LFAs, and the device can detect SARS-CoV-2-positive patient saliva samples missed by commercial LFAs. The technology provides an effective and practical solution to enhance the performance of conventional LFAs and will enable sensitive self-testing to prevent virus transmission and future outbreaks of new variants.

An autonomous microchip for real-time, label-free immune cell analysis.

Characterization of cell populations and identification of distinct subtypes based on surface markers are needed in a variety of applications from basic research and clinical assays to cell manufacturing. Conventional immunophenotyping techniques such as flow cytometry or fluorescence microscopy require immunolabeling of cells, expensive and complex instrumentation, skilled operators, and are therefore incompatible with field deployment and automated cell manufacturing systems. In this work, we introduce an autonomous microchip that can electronically quantify the immunophenotypical composition of a cell suspension. Our microchip identifies different cell subtypes by capturing each in different microfluidic chambers functionalized against the markers of the target populations. All on-chip activity is electronically monitored by an integrated sensor network, which informs an algorithm determining subpopulation fractions from chip-wide immunocapture statistics in real time. Moreover, optimal operational conditions within the chip are enforced through a closed-loop feedback control on the sensor data and the cell flow speed, and hence, the antibody-antigen interaction time is maintained within its optimal range for selective immunocapture. We apply our microchip to analyze a mixture of unlabeled CD4+ and CD8+ T cell sub-populations and then validated the results against flow cytometry measurements. The demonstrated capability to quantitatively analyze immune cells with no labels has the potential to enable not only autonomous biochip-based immunoassays for remote testing but also cell manufacturing bioreactors with built-in, adaptive quality control.

High throughput, label-free isolation of circulating tumor cell clusters in meshed microwells.

Extremely rare circulating tumor cell (CTC) clusters are both increasingly appreciated as highly metastatic precursors and virtually unexplored. Technologies are primarily designed to detect single CTCs and often fail to account for the fragility of clusters or to leverage cluster-specific markers for higher sensitivity. Meanwhile, the few technologies targeting CTC clusters lack scalability. Here, we introduce the Cluster-Wells, which combines the speed and practicality of membrane filtration with the sensitive and deterministic screening afforded by microfluidic chips. The >100,000 microwells in the Cluster-Wells physically arrest CTC clusters in unprocessed whole blood, gently isolating virtually all clusters at a throughput of >25 mL/h, and allow viable clusters to be retrieved from the device. Using the Cluster-Wells, we isolated CTC clusters ranging from 2 to 100+ cells from prostate and ovarian cancer patients and analyzed a subset using RNA sequencing. Routine isolation of CTC clusters will democratize research on their utility in managing cancer.

Immunomagnetic leukocyte differential in whole blood on an electronic microdevice.

Leukocytes are the frontline defense mechanism of the immune system. Their composition dynamically changes as a response to a foreign body, infection, inflammation, or other malignant behavior occurring within the body. Monitoring the composition of leukocytes, namely leukocyte differential, is a crucial assay periodically performed to diagnose an infection or to assess a person's vulnerability for a health anomaly. Currently, leukocyte differential analysis is performed using hematology analyzers or flow cytometers, both of which are bulky instruments that require trained and certified personnel for operation. In this work, we demonstrate a new technique to obtain leukocyte differentials in a highly portable and integrated microfluidic chip by magnetically analyzing the CD33 expression of leukocytes. When benchmarked against conventional laboratory instruments, our technology demonstrated <5% difference on average for all subtypes. Our results show that hematology testing could be performed beyond the centralized laboratories at a low cost and ultimately provide point-of-care and at-home testing opportunities.

Electronic measurement of cell antigen expression in whole blood.

Membrane antigens are phenotypic signatures of cells used for distinguishing various subpopulations and, therefore, are of great interest for diagnosis of diseases and monitoring of patients in hematology and oncology. Existing methods to measure antigen expression of a target subpopulation in blood samples require labor-intensive lysis of contaminating cells and subsequent analysis with complex and bulky instruments in specialized laboratories. To address this long-standing limitation in clinical cytometry, we introduce a microchip-based technique that can directly measure surface expression of target cells in hematological samples. Our microchip isolates an immunomagnetically-labeled target cell population from the contaminating background in whole blood and then utilizes the differential responses of target cells to on-chip magnetic manipulation to estimate their antigen expression. Moreover, manipulating cells with chip-sized permanent magnets and performing quantitative measurements via an on-chip electrical sensor network allows the assay to be performed in a portable platform with no reliance on laboratory infrastructure. Using our technique, we could successfully measure expressions of the CD45 antigen that is commonly expressed by white blood cells, as well as CD34 that is expressed by scarce hematopoietic progenitor cells, which constitutes only ∼0.0001% of all blood cells, directly from whole blood. With our technology, flow cytometry can potentially become a rapid bedside or at-home testing method that is available around the clock in environments where this invaluable assay with proven clinical utility is currently either outsourced or not even accessible.

Capillary flow control in lateral flow assays via delaminating timers.

Lateral flow assays (LFAs) use capillary flow of liquids for simple detection of analytes. While useful for spontaneously wicking samples, the capillary flow inherently limits performing complex reactions that require timely application of multiple solutions. Here, we introduce a technique to control capillary flow on paper by imprinting roadblocks on the flow path with water-insoluble ink and using the gradual formation of a void between a wetted paper and a sheath polymer tape to create timers. Timers are drawn at strategic nodes to hold the capillary flow for a desired period and thereby enable multiple liquids to be introduced into multistep chemical reactions following a programmed sequence. Using our technique, we developed (i) an LFA with built-in signal amplification to detect human chorionic gonadotropin with an order of magnitude higher sensitivity than the conventional assay and (ii) a device to extract DNA from bodily fluids without relying on laboratory instruments.

Negative enrichment of circulating tumor cells from unmanipulated whole blood with a 3D printed device.

Reliable and routine isolation of circulating tumor cells (CTCs) from peripheral blood would allow effective monitoring of the disease and guide the development of personalized treatments. Negative enrichment of CTCs by depleting normal blood cells ensures against a biased selection of a subpopulation and allows the assay to be applied on different tumor types. Here, we report an additively manufactured microfluidic device that can negatively enrich viable CTCs from clinically-relevant volumes of unmanipulated whole blood samples. Our device depletes nucleated blood cells based on their surface antigens and the smaller anucleated cells based on their size. Enriched CTCs are made available off the device in suspension making our technique compatible with standard immunocytochemical, molecular and functional assays. Our device could achieve a ~ 2.34-log depletion by capturing > 99.5% of white blood cells from 10 mL of whole blood while recovering > 90% of spiked tumor cells. Furthermore, we demonstrated the capability of the device to isolate CTCs from blood samples collected from patients (n = 15) with prostate and pancreatic cancers in a pilot study. A universal CTC assay that can differentiate tumor cells from normal blood cells with the specificity of clinically established membrane antigens yet require no label has the potential to enable routine blood-based tumor biopsies at the point-of-care.

Circulating Tumor Cell Enrichment Technologies.

Circulating tumor cells (CTCs) are responsible for the metastatic spread of cancer and therefore are extremely valuable not only for basic research on cancer metastasis but also as potential biomarkers in diagnosing and managing cancer in the clinic. While relatively non-invasive access to the blood tissue presents an opportunity, CTCs are mixed with approximately billion-times more-populated blood cells in circulation. Therefore, the accuracy of technologies for reliable enrichment of the rare CTC population from blood samples is critical to the success of downstream analyses. The focus of this chapter is to provide the reader an overview of significant advances made in the development of diverse CTC enrichment technologies by presenting the strengths of individual techniques in addition to specific challenges remaining to be addressed.

Combinatorial Immunophenotyping of Cell Populations with an Electronic Antibody Microarray.

Immunophenotyping is widely used to characterize cell populations in basic research and to diagnose diseases from surface biomarkers in the clinic. This process usually requires complex instruments such as flow cytometers or fluorescence microscopes, which are typically housed in centralized laboratories. Microfluidics are combined with an integrated electrical sensor network to create an antibody microarray for label-free cell immunophenotyping against multiple antigens. The device works by fractionating the sample via capturing target subpopulations in an array of microfluidic chambers functionalized against different antigens and by electrically quantifying the cell capture statistics through a network of code-multiplexed electrical sensors. Through a combinatorial arrangement of antibody sequences along different microfluidic paths, the device can measure the prevalence of different cell subpopulations in a sample from computational analysis of the electrical output signal. The device performance is characterized by analyzing heterogeneous samples of mixed tumor cell populations and then the technique is applied to determine leukocyte subpopulations in blood samples and the results are validated against complete blood cell count and flow cytometry results. Label-free immunophenotyping of cell populations against multiple targets on a disposable electronic chip presents opportunities in global health and telemedicine applications for cell-based diagnostics and health monitoring.

Hybrid negative enrichment of circulating tumor cells from whole blood in a 3D-printed monolithic device.

Isolation and analysis of circulating tumor cells (CTCs) from blood samples present exciting opportunities for basic cancer research and personalized treatment of the disease. While microchip-based negative CTC enrichment offers both sensitive microfluidic cell screening and unbiased selection, conventional microchips are inherently limited by their capacity to deplete a large number of normal blood cells. In this paper, we use 3D printing to create a monolithic device that combines immunoaffinity-based microfluidic cell capture and a commercial membrane filter for negative enrichment of CTCs directly from whole blood. In our device, stacked layers of chemically-functionalized microfluidic channels capture millions of white blood cells (WBCs) in parallel without getting saturated and the leuko-depleted blood is post-filtered with a 3 µm-pore size membrane filter to eliminate anucleated blood cells. This hybrid negative enrichment approach facilitated direct extraction of viable CTCs off the chip on a membrane filter for downstream analysis. Immunofluorescence imaging of enriched cells showed ∼90% tumor cell recovery rate from simulated samples spiked with prostate, breast or ovarian cancer cells. We also demonstrated the feasibility of our approach for processing clinical samples by isolating prostate cancer CTCs directly from a 10 mL whole blood sample.

Electronic profiling of membrane antigen expression via immunomagnetic cell manipulation.

Membrane antigens control cell function by regulating biochemical interactions and hence are routinely used as diagnostic and prognostic targets in biomedicine. Fluorescent labeling and subsequent optical interrogation of cell membrane antigens, while highly effective, limit expression profiling to centralized facilities that can afford and operate complex instrumentation. Here, we introduce a cytometry technique that computes surface expression of immunomagnetically labeled cells by electrically tracking their trajectory under a magnetic field gradient on a microfluidic chip with a throughput of >500 cells per min. In addition to enabling the creation of a frugal cytometry platform, this immunomagnetic cell manipulation-based measurement approach allows direct expression profiling of target subpopulations from non-purified samples. We applied our technology to measure epithelial cell adhesion molecule expression on human breast cancer cells. Once calibrated, surface expression and size measurements match remarkably well with fluorescence-based measurements from a commercial flow cytometer. Quantitative measurements of biochemical and biophysical cell characteristics with a disposable cytometer have the potential to impact point of care testing of clinical samples particularly in resource limited settings.

Photodynamic Inactivation of Multidrug-Resistant Staphylococcus aureus Using Hybrid Photosensitizers Based on Amphiphilic Block Copolymer-Functionalized Gold Nanoparticles.

Multidrug-resistant Staphylococcus aureus (MRSA) has become one of the major causes of various infections, leading to morbidity in both healthy and immune-compromised populations worldwide. Herein, we report a novel type of hybrid photosensitizer based on amphiphilic block copolymer-functionalized gold nanoparticles. The design of the nanoparticles provides a facile means to incorporate hydrophobic photosensitizing molecules for use in aqueous media. The hybrid photosensitizers display greatly enhanced singlet oxygen generation and outstanding photodynamic inactivation (PDI) efficacy against MRSA under light illumination. These hybrid photosensitizers greatly improve the effectiveness of PDI against MRSA while not involving antibiotics.

Magnetic relaxation-based sensing of phosphate ion.

We report a novel magnetic relaxation-based sensing method for sensitive and selective detection of phosphate ions in aqueous media using paramagnetic nanoparticles. The method can detect phosphate ions at physiological pH quantitatively with high selectivity, even in a commercial fertilizer without separation.