Scalable cryopreservation of infectious Cryptosporidium hominis oocysts by vitrification.

Cryptosporidium hominis is a serious cause of childhood diarrhea in developing countries. The development of therapeutics is impeded by major technical roadblocks including lack of cryopreservation and simple culturing methods. This impacts the availability of optimized/standardized singular sources of infectious parasite oocysts for research and human challenge studies. The human C. hominis TU502 isolate is currently propagated in gnotobiotic piglets in only one laboratory, which limits access to oocysts. Streamlined cryopreservation could enable creation of a biobank to serve as an oocyst source for research and distribution to other investigators requiring C. hominis. Here, we report cryopreservation of C. hominis TU502 oocysts by vitrification using specially designed specimen containers scaled to 100 µL volume. Thawed oocysts exhibit ~70% viability with robust excystation and 100% infection rate in gnotobiotic piglets. The availability of optimized/standardized sources of oocysts may streamline drug and vaccine evaluation by enabling wider access to biological specimens.

Point-of-care semi-quantitative test for adherence to tenofovir alafenamide or tenofovir disoproxil fumarate.

OBJECTIVES: Objective measurement of antiretrovirals may aid clinical interventions for improving adherence to HIV prevention or treatment regimens. A point-of-care urine test could provide real-time information about recent adherence to regimens containing tenofovir disoproxil fumarate or tenofovir alafenamide. We developed a lateral flow immunoassay (LFA) and ELISA for urinary tenofovir. METHODS: The intensity of the LFA test line was quantified using an optical reader and visually scored 0-5 by two independent people, using a reference card. The sensitivity and specificity of both the ELISA and LFA were determined for two different tenofovir concentration cut-offs for tenofovir disoproxil fumarate and tenofovir alafenamide adherence-1500 and 150 ng/mL, respectively. To validate the assays, we measured 586 urine samples from 28 individuals collected as part of a study of tenofovir pharmacokinetics in adults, which were also measured by MS for reference. RESULTS: Both the LFA signal and ELISA signal were each strongly correlated with drug concentrations (0.91 and 0.92, respectively). The LFA signal and ELISA were highly sensitive and specific at both thresholds (LFA sensitivity/specificity: tenofovir disoproxil fumarate, 89%/96%; and tenofovir alafenamide, 90%/96%) (ELISA sensitivity/specificity: tenofovir disoproxil fumarate, 94%/94%; and tenofovir alafenamide, 92%/84%). Visual scoring of the LFA was also highly sensitive and specific at both the tenofovir disoproxil fumarate threshold and the tenofovir alafenamide threshold (sensitivity/specificity: tenofovir disoproxil fumarate, 91%/94%; and tenofovir alafenamide, 87%/90%). CONCLUSIONS: Our rapid semi-quantitative test can measure tenofovir concentrations relevant to both tenofovir alafenamide and tenofovir disoproxil fumarate adherence, which may support adherence-promoting interventions across a range of HIV care settings.

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events.

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

Slow-delivery and distributed exchange of cryoprotective agents with hydrogel beads.

Intracellular loading of cryoprotective agents (CPAs) into target cells is a critical step for cryopreservation. However, biological membranes are usually much less permeable to CPAs than to water, resulting in high osmotic pressures and osmotic damage during the CPA loading and unloading phases of cryopreservation. Here, we show that calcium alginate hydrogel beads several millimeters in diamater containing CPAs can be admixed with a cell suspension to spontaneously release CPAs in a gradual and distributed manner. We demonstrate that beads containing cell media enable the gradual removal of CPA from Jurkat cells equilibrated in a typical cryopreservation solution of 15% glycerol, protecting the cells from hypotonic damage. We show that the dynamics of CPA exchange are accurately described by a numerical model of free diffusion within the gel. This approach may enable semiautomated and closed methods of gradual CPA exchange from large volume cell suspensions.

Point-of-care and Near Real-time Testing for Antiretroviral Adherence Monitoring to HIV Treatment and Prevention.

PURPOSE OF REVIEW: In this report, we review the need for point-of-care (POC) or near real-time testing for antiretrovirals, progress in the field, evidence for guiding implementation of these tests globally, and future directions in objective antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) adherence monitoring. RECENT FINDINGS: Two cornerstones to end the HIV/AIDS pandemic are ART, which provides individual clinical benefits and eliminates forward transmission, and PrEP, which prevents HIV acquisition with high effectiveness. Maximizing the individual and public health benefits of these powerful biomedical tools requires high and sustained antiretroviral adherence. Routine monitoring of medication adherence in individuals receiving ART and PrEP may be an important component in interpreting outcomes and supporting optimal adherence. Existing practices and subjective metrics for adherence monitoring are often inaccurate or unreliable and, therefore, are generally ineffective for improving adherence. Laboratory measures of antiretroviral concentrations using liquid chromatography tandem mass spectrometry have been utilized in research settings to assess medication adherence, although these are too costly and resource-intensive for routine use. Newer, less costly technologies such as antibody-based methods can provide objective drug-level measurement and may allow for POC or near-patient adherence monitoring in clinical settings. When coupled with timely and targeted counseling, POC drug-level measures can support adherence clinic-based interventions to ART or PrEP in near real time.

Robust Visualization and Discrimination of Nanoparticles by Interferometric Imaging.

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.

High throughput intracellular delivery by viscoelastic mechanoporation.

Brief pulses of electric field (electroporation) and/or tensile stress (mechanoporation) have been used to reversibly permeabilize the plasma membrane of mammalian cells and deliver materials to the cytosol. However, electroporation can be harmful to cells, while efficient mechanoporation strategies have not been scalable due to the use of narrow constrictions or needles which are susceptible to clogging. Here we report a high throughput approach to mechanoporation in which the plasma membrane is stretched and reversibly permeabilized by viscoelastic fluid forces within a microfluidic chip without surface contact. Biomolecules are delivered directly to the cytosol within seconds at a throughput exceeding 250 million cells per minute. Viscoelastic mechanoporation is compatible with a variety of biomolecules including proteins, RNA, and CRISPR-Cas9 ribonucleoprotein complexes, as well as a range of cell types including HEK293T cells and primary T cells. Altogether, viscoelastic mechanoporation appears feasible for contact-free permeabilization and delivery of biomolecules to mammalian cells ex vivo.

High throughput intracellular delivery by viscoelastic mechanoporation.

Brief and intense electric fields (electroporation) and/or tensile stresses (mechanoporation) have been used to temporarily permeabilize the plasma membrane of mammalian cells for the purpose of delivering materials to the cytosol. However, electroporation can be harmful to cells, while efficient mechanoporation strategies have not been scalable due to the use of narrow constrictions or needles which are susceptible to clogging. Here we report a method of mechanoporation in which cells were stretched and permeabilized by viscoelastic flow forces without surface contact. Inertio-elastic cell focusing aligned cells to the center of the device, avoiding direct contact with walls and enabling efficient (95%) intracellular delivery to over 200 million cells per minute. Functional biomolecules such as proteins, RNA, and ribonucleoprotein complexes were successfully delivered to Jurkat cells. Efficient intracellular delivery to HEK293T cells and primary activated T cells was also demonstrated. Contact-free mechanoporation using viscoelastic fluid forces appears to be feasible method for efficient and high throughput intracellular delivery of biomolecules to mammalian cells ex vivo.

Rapid prototyping for high-pressure microfluidics.

Soft lithography has permitted rapid prototyping of precise microfluidic features by patterning a deformable elastomer such as polydimethylsiloxane (PDMS) with a photolithographically patterned mold. In microfluidics applications where the flexibility of PDMS is a drawback, a variety of more rigid materials have been proposed. Compared to alternatives, devices fabricated from epoxy and glass have superior mechanical performance, feature resolution, and solvent compatibility. Here we provide a detailed step-by-step method for fabricating rigid microfluidic devices from soft lithography patterned epoxy and glass. The bonding protocol was optimized yielding devices that withstand pressures exceeding 500 psi. Using this method, we demonstrate the use of rigid high aspect ratio spiral microchannels for high throughput cell focusing.

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.

Attomolar sensitivity microRNA detection using real-time digital microarrays.

MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA-451a (miR-451) and miRNA-223-3p (miR-223). We demonstrated improvements in sensitivity in comparison to traditional end-point assays that employ capture on solid phase support, by implementing real-time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low-abundance biomarkers in the presence of low-affinity but high-abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to [Formula: see text] 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.

Cryopreservation of infectious Cryptosporidium parvum oocysts achieved through vitrification using high aspect ratio specimen containers.

Infection with protozoa of the genus Cryptosporidium is a leading cause of child morbidity and mortality associated with diarrhea in the developing world. Research on this parasite has been impeded by many technical limitations, including the lack of cryopreservation methods. While cryopreservation of Cryptosporidium oocysts by vitrification was recently achieved, the method is restricted to small sample volumes, thereby limiting widespread implementation of this procedure. Here, a second-generation method is described for cryopreservation of C. parvum oocysts by vitrification using custom high aspect ratio specimen containers, which enable a 100-fold increase in sample volume compared to previous methods. Oocysts cryopreserved using the described protocol exhibit high viability, maintain in vitro infectivity, and are infectious to interferon-gamma (IFN-γ) knockout mice. Importantly, the course of the infection is comparable to that observed in mice infected with unfrozen oocysts. Vitrification of C. parvum oocysts in larger volumes will expedite progress of research by enabling the sharing of isolates among different laboratories and the standardization of clinical trials.

Immunoassay for HIV Drug Metabolites Tenofovir and Tenofovir Diphosphate.

Poor patient adherence to antiretroviral medication represents a major obstacle for managing disease and reducing rates of new HIV infections. The measurement of patient drug levels is the most objective method of determining adherence. Tenofovir and tenofovir diphosphate are metabolites of some of the most common HIV medications for treatment and prevention and can be quantified by mass spectrometry. Here, we report the development of a competitive enzyme linked immunoassay as a simplified approach for detecting tenofovir and tenofovir diphosphate. Monoclonal antibodies were produced by two tenofovir-hapten conjugates and screened for binding to immobilized tenofovir, and then for competition by tenofovir and tenofovir diphosphate. Antibody specificity was evaluated against adenosine phosphates, which are close structural analogs. We performed numerical simulations of reaction equilibrium to guide assay optimization. When used to evaluate spiked tenofovir in plasma and spiked tenofovir diphosphate in red blood cell lysate, the optimized assay had high sensitivity and specificity.

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.

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels.

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique's simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.

Quantitative interferometric reflectance imaging for the detection and measurement of biological nanoparticles.

The sensitive detection and quantitative measurement of biological nanoparticles such as viruses or exosomes is of growing importance in biology and medicine since these structures are implicated in many biological processes and diseases. Interferometric reflectance imaging is a label-free optical biosensing method which can directly detect individual biological nanoparticles when they are immobilized onto a protein microarray. Previous efforts to infer bio-nanoparticle size and shape have relied on empirical calibration using a 'ruler' of particle samples of known size, which was inconsistent and qualitative. Here, we present a mechanistic physical explanation and experimental approach by which interferometric reflectance imaging may be used to not only detect but also quantitatively measure bio-nanoparticle size and shape. We introduce a comprehensive optical model that can quantitatively simulate the scattering of arbitrarily-shaped nanoparticles such as rod-shaped or filamentous virions. Finally, we optimize the optical design for the detection and quantitative measurement of small and low-index bio-nanoparticles immersed in water.

Real-Time Capture and Visualization of Individual Viruses in Complex Media.

Label-free imaging of individual viruses and nanoparticles directly in complex solutions is important for virology research and biosensing applications. A successful visualization technique should be rapid, sensitive, and inexpensive, while needing minimal sample preparation or user expertise. Current approaches typically require fluorescent labeling or the use of an electron microscope, which are expensive and time-consuming to use. We have developed an imaging technique for real-time, sensitive, and label-free visualization of viruses and nanoparticles directly in complex solutions such as serum. By combining the advantages of a single-particle reflectance imaging sensor, with microfluidics, we perform real-time digital detection of individual 100 nm vesicular stomatitis viruses as they bind to an antibody microarray. Using this approach, we have shown capture and visualization of a recombinant vesicular stomatitis virus Ebola model (rVSV-ZEBOV) at 100 PFU/mL in undiluted fetal bovine serum in less than 30 min.

Spatial periodicity in growth plate shear mechanical properties is disrupted by vitamin D deficiency.

The growth plate is a highly organized section of cartilage in the long bones of growing children that is susceptible to mechanical failure as well as structural and functional disruption caused by a dietary deficiency of vitamin D. The shear mechanical properties of the proximal tibial growth plate of rats raised either on normal or vitamin D and calcium deficient diets were measured. A sinusoidal oscillating shear load was applied to small excised growth plate specimens perpendicular to the direction of growth while imaging the deformation in real time with a fast confocal microscope. Local deformations and shear strains were quantified using image correlation. The proliferative zone of the growth plate bores the majority of the shear strain and the resting, hypertrophic and calcification zones deformed less. Surprisingly, we regularly observed discontinuous deformations in the proliferative zone in both groups that resembled cell columns sliding past one another in the direction of growth. These discontinuities manifested as regions of concentrated longitudinal shear strain. Furthermore, these shear strain concentrations were spaced evenly in the proliferative zone and the spacing between them was similar across growth plate regions and across control specimens. In contrast to the healthy controls, the vitamin D deficient growth plate exhibited larger variations in the size and orientation of cellular columns in the proliferative and hypertrophic zones. High strains were observed between columns, much as they were in the controls. However, the regular spacing of shear strain concentrations was not preserved, echoing the observation of decreased structural organization.