Browsing by Subject "Point-of-care"

Bioanalytical techniques such as immunoassays are ubiquitous in clinical and basic research laboratories and have transformed how we diagnose patients, monitor health, and study disease. Immunoassays typically use capture reagents, such as antibodies or antigens, to detect and quantify a biomarker of interest from a clinical specimen based on highly sensitive and specific binding interactions. While laboratory-based assays, such as enzyme-linked immunosorbent assay (ELISA), are the workhorses of clinical laboratories, they have several shortcomings that limit their overall utility, especially in low resource settings. Of note, ELISA requires multiple timed incubation steps, trained personnel, expensive equipment, and suffers from long times to result. To democratize access to clinical-grade tests, researchers have sought out different methods for point-of-care (POC) testing that are easy to perform and maintain high sensitivity and specificity. This dissertation describes the use of a “zero-background” polymer coating—poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)—as a substrate for highly sensitive and specific POC diagnostic tests. The POEGMA coating eliminates nearly all non-specific protein adsorption and cellular adhesion, thus leading to high signal-to-noise ratios, even from complex biological samples, such as whole blood. In addition, the POEGMA brush contains all biomolecules necessary to complete an assay after addition of a liquid sample, thus allowing assays to be conducted in a single step. Further, the POEGMA coating stabilizes biomolecules on the surface, which allows tests to be stored at ambient conditions without refrigeration. Assays are read out using a fluorescence detector which quantifies the concentration for a given biomarker of interest. By inkjet printing capture biomolecules at discrete spatial addresses on the POEGMA-slides, multiplexing can be accomplished using a single fluorophore which greatly reduces the complexity and costs for assay readout. This dissertation focuses on adapting and applying this platform to several clinically relevant applications. First, we developed a test for molecular and cellular credentialing of breast cancer tissue at the POC (Chapter 2). With the onset of the coronavirus 2019 (COVID-19) pandemic, we adapted the platform to detect several different relevant biomarkers for COVID-19, including total antibody concentration against several viral proteins (Chapter 3), neutralizing antibodies (Chapter 4), and viral proteins (Chapter 5). All the tests developed for COVID-19 use multiplexed sensing strategies and can be conducted with minimal/no user intervention or clinical infrastructure. Taken together, these studies highlight the great potential for bioanalytical assays built upon POEGMA-coated substrates to be used for clinical applications in disease diagnosis, surveillance, and management.

Aims: Measurement of glycated hemoglobin (HbA1c) is an important indicator of glucose control over time. Point-of-care (POC) devices allow for rapid and convenient measurement of HbA1c, greatly facilitating diabetes care. We assessed two POC analyzers in the Peruvian Amazon where laboratory-based HbA1c testing is not available.

Methods: Venous blood samples were collected from 203 individuals from six different Amazonian communities with a wide range of HbA1c, 4.4-9.0% (25-75 mmol/mol). The results of the Afinion AS100 and the DCA Vantage POC analyzers were compared to a central laboratory using the Premier Hb9210 high-performance liquid chromatography (HPLC) method. Imprecision was assessed by performing 14 successive tests of a single blood sample.

Results: The correlation coefficient r for POC and HPLC results was 0.92 for the Afinion and 0.93 for the DCA Vantage. The Afinion generated higher HbA1c results than the HPLC (mean difference = +0.56% [+6 mmol/mol]; p < 0.001), as did the DCA Vantage (mean difference = +0.32% [4 mmol/mol]). The bias observed between POC and HPLC did not vary by HbA1c level for the DCA Vantage (p = 0.190), but it did for the Afinion (p < 0.001). Imprecision results were: CV = 1.75% for the Afinion, CV = 4.01% for the DCA Vantage. Sensitivity was 100% for both devices, specificity was 48.3% for the Afinion and 85.1% for the DCA Vantage, positive predictive value (PPV) was 14.4% for the Afinion and 34.9% for the DCA Vantage, and negative predictive value (NPV) for both devices was 100%. The area under the receiver operating characteristic (ROC) curve was 0.966 for the Afinion and 0.982 for the DCA Vantage. Agreement between HPLC and POC in classifying diabetes and prediabetes status was slight for the Afinion (Kappa = 0.12) and significantly different (McNemar’s statistic = 89; p < 0.001), and moderate for the DCA Vantage (Kappa = 0.45) and significantly different (McNemar’s statistic = 28; p < 0.001).

Conclusions: Despite significant variation of HbA1c results between the Afinion and DCA Vantage analyzers compared to HPLC, we conclude that both analyzers should be considered in health clinics in the Peruvian Amazon for therapeutic adjustments if healthcare workers are aware of the differences relative to testing in a clinical laboratory. However, imprecision and bias were not low enough to recommend either device for screening purposes, and the local prevalence of anemia and malaria may interfere with diagnostic determinations for a substantial portion of the population.

As the cost of medical care increases, people are relying increasingly on internet diagnosis and community care rather than the expertise of medical professionals. Technological and medical advances have facilitated a partial answer through the increase in handheld sensing apparatuses. Yet even with these developments, significant further advancements are required to further drive down fabrication requirements (both in terms of cost and environmental impact) and facilitate fully-integrated and easy to use sensors. Printing electronics could be a powerful tool to accomplish this as printing allows for low-cost fabrication of high-area electronics. The vast majority of printed electronics reports focus on utilization of already developed commercial inks to create devices with new functionalities. This significantly limits development because current inks both necessitate damaging post processing—which precludes the use of delicate substrates, making skin-integration impossible—and many inks require bespoke printing processes, which increases fabrication complexity and thus cost. Further, with the proliferation of single-use medical testing, consideration must be made towards environmental compatibility. Therefore, innovations in electronic ink formulation and printing geared towards addressing the post-processing and environmental impact concerns are needed to enable continued progress towards printed POC sensors. The work contained in this dissertation centers around the development of inks intended to advance electronic biosensing applications. Focus is on using aerosol jet printing to enable the printing of nanomaterials and utilizes the unique properties of these nanomaterials—such as functionality immediately after printing, recyclability, and compatibility with deposition directly on biological surfaces (i.e., human skin)—to develop technologies intended to democratize healthcare. Notably, low temperature printable silver nanowire (AgNW) inks for the creation of biologically integrated electronics are demonstrated. Electrically conductive inks are created that are capable of achieving high conductivities when directly deposited onto living tissue at temperatures compatible with life (20 °C). The conductive lines yielded high resistance to degradation from bending strain, with a mere 8% decrease in conductivity when the plastic film on which they were printed was folded in half. As a demonstration, the AgNW ink was printed onto a human finger and used to illuminate a small light that remained illuminated even when the finger was bent. These results pave the way towards patient-specific medical diagnostics that are comfortable to wear, easy to use, and designed towards the needs of each individual patient. Next, a printing method to deposit biological sensing proteins for biomedical assays is investigated. Traditional techniques require extended time and the use of large quantities of immensely expensive proteins to make biosensors. Herein, a decade-old belief that aerosol jet printing is incompatible with the deposition of proteins is overturned, and, in doing so, highly sensitive biosensors for carcinoembryonic antigen (CEA) that compare favorably the mainstay fabrication technique that is known to impart no damage to the printed biological inks is demonstrated. Finally, the co-printing of a bio-recognition element with the previously mentioned electrically conductive AgNW ink demonstrate the potential for the future investigation of a fully aerosol-jet printed electronic biosensor. To address the environmental waste accumulation concern that plagues the advancement of ubiquitous patient-guided sensing, inks that facilitate the creation of fully-printed, all-carbon recyclable electronics (ACRE) are investigated. The combination of nanocrystalline cellulose, graphene and semiconducting carbon nanotubes enable the first fully recyclable transistor device. The ACRE transistors maintain high stability for over 10 months, display among the best performance of any printed transistor (Ion/Ioff: 104 and Ion 65 µA µm-1) and can be entirely deconstructed for recapture and reuse of the constituent CNT and graphene inks with near 100% nanomaterial retention and the biodegradation of the cellulose-based components. ACRE-based lactate sensors are used as an illustration of utility to show the versatility of the platform. Finally, as a culminating demonstration, a fully-printed chip for the handheld measurement of blood clot time (prothrombin time) was developed. Printing the entirety of the device allows for the creation of a low-cost chip for the simple, fast, and robust measurement of human blood clot times. In addition, a custom-designed, handheld control system with a 3D-printed case was developed to create a fully integrated point-of-care measurement platform towards simplifying medicine dosing strategies. The work described herein marks a significant leap in the development of printed inks to enable custom biological sensing applications. Once fully realized, these applications will mark a watershed, ushering in an era of individualized medicine with ubiquitous sensing to actively track disease progression in real-time. We are at the dawn of a new era in medicine that focuses more on prevention and control as opposed to reaction. One future direction for this work is promoting directly printed and reusable on-skin theragnostics for bespoke patient care such as the delivery and monitoring of pain medication that allows for better oversite over use and misuse.

Fluorescence-based methodologies have been used extensively for biosensing and to analyze molecular dynamics and interactions. An emerging, promising diagnostic tool are fluorescence-based microarrays due to their high throughput, small sample volume and multiplexing capabilities. However, their low fluorescence output has limited their implementation for in vitro diagnostics applications in a point-of-care (POC) setting. Here, by integration of a sandwich immunoassay microarray within a plasmonic nanogap metasurface, we demonstrate strongly enhanced fluorescence enabling readout by a fluorescence microarray even at low sensitivities. We have named this plasmonic architecture the plasmonically enhanced D4 (PED4) assay. The immunoassay consists of inkjet-printed capture and fluorescently labeled detection antibodies on a polymer brush which is grown on a gold film. Colloidally synthesized silver nanocubes (SNCs) are placed on top of the brush through a polyelectrolyte layer and interacts with the underlying gold film creating a nanogap plasmonic structure supporting local electromagnetic field enhancements of ~100-fold. By varying the thickness of the brush between 5 and 20 nm, a 151-fold increase in fluorescence and a 14-fold improvement in the limit-of-detection (LOD) is observed for the cardiac biomarker B-type natriuretic peptide (BNP) compared to the unenhanced assay, paving the way for a new generation of point-of-care clinical diagnostics.

To move the PED4 towards a single step point of care test (POCT), SNCs are conjugated with a secondary antibody that attaches specifically to the detection antibody. This allows SNCs to deposit on the surface without the need of a charged polyelectrolyte layer. In addition, multiplexing capabilities are demonstrated in this plasmonic platform where NT-proBNP, Galectin-3, and NGAL are simultaneously detected and fluorescently enhanced. Microfluidics integration and use of a low-cost detector is also demonstrated.

Microfluidic technologies, and the subset of devices that integrate acoustics into their designs (known as acoustofluidic devices), present great potential for solving the challenges of the future. One specific subset of these technologies, termed sharp-edge based acoustofluidics, has shown promise in a variety applications; specifically, previous work has explored the use of this technology in applications such as fluid pumping and mixing, cell stimulation, and bio-sample preparation. However, even though there are a vast number of applications that sharp-edge based acoustofluidics have been applied to, there are several shortcomings that need to be addressed.

First, and perhaps most critically, very little is known about the fundamental mechanism of this platform’s operation. The search for novel applications has left a gap in the knowledge base for understanding how these devices work on a fundamental level; gaining a better understanding of how the technology works may open the door to finding new and previously unimagined applications. Second, although not a problem that is specifically limited to sharp-edge based acoustofluidic devices, the technology suffers from serious limitations in real world applicability. That is, even though these devices have advantages over traditional techniques, including speed, cost, and ease of use, they are unable to be taken advantage of. For this reason, there is a critical need to demonstrate a viable pathway to real-world usage.

In an attempt to tackle these shortcomings, we begin our research by investigating the vibrational profile generated within a sharp-edge mixer. Throughout this exploration we uncover that the mechanism behind the technology’s success is relatively low frequency flexural waves which have wavelengths commensurate with the overall dimensions of the technology. This is in contrast to the previous belief that waves with lengths many times larger than the device itself were dominating; as a result, we developed and explored a novel platform for particle manipulation based on wave interference (not unlike high frequency based acoustofluidic platforms). This technology offers a new technique for interacting with micro particles and cells in an open fluid chamber. In order to improve the technology’s adoptability, we also developed and characterized two unique and portable control platforms towards eventual point-of-care (POC) use.

Altogether, this work serves to further the knowledge and relevance of sharp-edge based technology. It is our hope that this work can serve as a starting point for future explorations into novel platforms which make use of the small wavelength vibrations achievable with this low cost setup. Additionally, we hope that this work may motivate the broader field to transition their technology into equally accessible platforms, such that the microfluidics community as a whole can bring their useful technology to practical applications.