Amy E. Herr

Microfluidic immunoassays as rapid saliva-based clinical diagnostics

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 μl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids.

Single-cell western blotting

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼103 concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.

Saliva/Pathogen Biomarker Signatures and Periodontal Disease Progression

The purpose of this study was to determine the role of saliva-derived biomarkers and periodontal pathogens during periodontal disease progression (PDP). One hundred human participants were recruited into a 12-month investigation. They were seen bi-monthly for saliva and clinical measures and bi-annually for subtraction radiography, serum and plaque biofilm assessments. Saliva and serum were analyzed with protein arrays for 14 pro-inflammatory and bone turnover markers, while qPCR was used for detection of biofilm. A hierarchical clustering algorithm was used to group study participants based on clinical, microbiological, salivary/serum biomarkers, and PDP. Eighty-three individuals completed the six-month monitoring phase, with 44 exhibiting PDP, while 39 demonstrated stability. Participants assembled into three clusters based on periodontal pathogens, serum and salivary biomarkers. Cluster 1 members displayed high salivary biomarkers and biofilm; 82% of these individuals were undergoing PDP. Cluster 2 members displayed low biofilm and biomarker levels; 78% of these individuals were stable. Cluster 3 members were not discriminated by PDP status; however, cluster stratification followed groups 1 and 2 based on thresholds of salivary biomarkers and biofilm pathogens. The association of cluster membership to PDP was highly significant (p < 0.0002). The use of salivary and biofilm biomarkers offers potential for the identification of PDP or stability (ClinicalTrials.gov number, CT00277745).

Microfluidics: reframing biological enquiry

The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools — in conjunction with advanced imaging, bioinformatics and molecular biology approaches — will transform biology into a precision science.

Microfluidic Western blotting

Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10–60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.

Microfluidic integration for automated targeted proteomic assays

A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of protein isoforms would accelerate development of effective protein-based diagnostics. Toward scalable protein isoform analysis, we introduce a microfluidic “single-channel, multistage” immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting. In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (ca. 1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients. The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories.

Automated microfluidic protein immunoblotting

This protocol describes regional photopatterning of polyacrylamide gels in glass microfluidic devices as a platform for seamless integration of multiple assay steps. The technology enables rapid, automated protein immunoblotting, demonstrated in this study for native western blotting. The fabrication procedure is straightforward and requires approximately 3 h from the start of gel photopatterning to completion of native protein western blotting, a substantial time savings over slab-gel immunoblotting. The assay itself requires less than 5 min. Importantly, all assay stages are programmably controlled by a high-voltage power supply and monitored by an epifluorescence microscope equipped with a charge-coupled device camera. Our approach overcomes severe limitations associated with conventional immunoblotting, including multiple steps requiring manual intervention, low throughput and substantial consumption of reagents. We also describe a simple chemical recycling protocol so that glass chips can be reused. The fabrication technique described forms the basis for a diverse suite of bioanalytical tools, including DNA/RNA blotting and multidimensional separations.

Integrated microfluidic platform for oral diagnostics.

Abstract:  While many point‐of‐care (POC) diagnostic methods have been developed for blood‐borne analytes, development of saliva‐based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip‐based immunoassays (<3–10 min) with low sample volume requirements (10 μL) and appreciable sensitivity (nM–pM). Our microfluidic method facilitates hands‐free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid‐handling components, and data acquisition software to develop a portable, self‐contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip‐based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.

Microfluidic Polyacrylamide Gel Electrophoresis with in Situ Immunoblotting for Native Protein Analysis

We introduce an automated immunoblotting method that reports protein electrophoretic mobility and identity in a single streamlined microfluidic assay. Native polyacrylamide gel electrophoresis (PAGE) was integrated with subsequent in situ immunoblotting. Integration of three PA gel elements into a glass microfluidic chip achieved multiple functions, including (1) rapid protein separation via on-chip PAGE, (2) directed electrophoretic transfer of resolved protein peaks to an in-line blotting membrane, and (3) high-efficiency identification of the transferred proteins using antibody-functionalized blotting membranes. In-chip blotting membranes were photopatterned with biotinylated antibody using streptavidin polyacrylamide (PA) thus yielding postseparation sample analysis. No pressure driven flow or fluid valving was required, as the assay was operated by electrokinetically programmed control. A model sample of fluorescently labeled BSA (negative control), alpha-actinin, and prostate specific antigen (PSA) was selected to develop and characterize the assay. A 5 min assay time was required without operator intervention. Optimization of the blotting membrane (geometry, operation, and composition) yielded a detection limit of approximately 0.05 pg (alpha-actinin peak). An important additional blotting fabrication strategy was developed and characterized to allow vanishingly small antibody consumption (approximately 1 microg), as well as end-user customization of the blotting membrane after device fabrication and storage. This first report of rapid on-chip protein PAGE integrated with in situ immunoblotting forms the basis for a sensitive, automated approach applicable to numerous forms of immunoblotting.

Human Tear Protein Analysis Enabled by an Alkaline Microfluidic Homogeneous Immunoassay

The ability to probe the protein content of human tear fluid has enormous potential for deepening our understanding of ocular and systemic disease pathology and enabling novel noninvasive tear-based diagnostic technologies. To overcome current challenges in tear proteomic measurements, we report on the first microfluidic homogeneous immunoassay capable of making rapid, quantitative, and specific measurements of endogenous tear protein biomarkers in human tear fluid. Lactoferrin (Lf) is a tear-specific biomarker for Sjögrens syndrome (SS), a serious systemic autoimmune disease currently diagnosed through rudimentary volumetric and surface chemistry measurements and an invasive lip biopsy. We detail optimization of a homogeneous electrophoretic immunoassay for Lf in <1 μL of tear fluid at clinically relevant concentrations. In particular, we present assay development details and a final assay that enables quantification of Lf in <5 s in a clinically relevant range for SS diagnostics. Characterization suggests the on-chip assay is accurate to within 15% of ELISA, specific (<15% nonspecific signal), and with a lower limit of detection of 3 ± 2 nM Lf in human tear matrix. Additionally, we develop and characterize a protocol for eluting proteins from nitrocellulose Schirmer strips, the clinical de facto standard for tear collection and storage. We relate on-chip measured Lf concentrations back to ocular surface concentrations for the first time to our knowledge. Taken in sum, this work details important steps toward (1) expanding the set of proteins quantified by electrophoretic immunoassays to encompass a wider range of isoelectric points than has been reported, (2) creating a first-in-kind translatable assay with clinical relevance to SS diagnostics, and (3) expanding the analytical toolkit available for rapid tear protein measurements, as is relevant to the advancement of basic research and clinical medicine.