Colorimetric Detection of Fentanyl Powder on Surfaces Using a Supramolecular Displacement Assay.

Fentanyl is a potent synthetic opioid with an alarmingly low lethal dosage of 2 mg. The equipment necessary to detect fentanyl in field settings (e.g., hand-held spectrometers) is restricted to highly trained, well-funded, and specialized personnel. Established point-of-need technologies, such as lateral flow immunochromatographic strips, are available; however, they often involve multiple contact-based steps (e.g., collection, mixing) that pose a higher risk to users handling unknown substances. Herein, we developed a colorimetric displacement assay capable of contactless detection of fentanyl in liquid or solid samples. The basis of our assay relies on the presence of fentanyl to displace a redox mediator, ferrocene carboxylic acid, inclusively bound in the cavity of a supramolecular host, CB[7]. The displacement is only possible in the presence of high affinity binding guests, like fentanyl (KA ∼ 106 M-1). The liberated redox guest can then react with indicator reagents that are free in solution, producing either: (i) a distinct blue color to indicate the presence of fentanyl or (ii) a pale blue tint in the absence of fentanyl. We demonstrate rapid and specific detection of fentanyl free base and fentanyl derivatives (e.g., acetyl fentanyl and furanyl fentanyl) against a panel of 9 other common drugs of abuse (e.g., morphine, cocaine, and heroin). Furthermore, we highlight the intended use of this assay by testing grains of fentanyl derivatives on a surface with a drop (i.e., 25 µL) of the assay reagent. We anticipate that this approach can be applied broadly to identify the presence of fentanyl at the point of need.

Rapid spectrophotometric detection for optimized production of landomycins and characterization of their therapeutic potential.

Microbial-derived natural products remain a major source of structurally diverse bioactive compounds and chemical scaffolds that have the potential as new therapeutics to target drug-resistant pathogens and cancers. In particular, genome mining has revealed the vast number of cryptic or low-yield biosynthetic gene clusters in the genus Streptomyces. However, low natural product yields-improvements to which have been hindered by the lack of high throughput methods-have slowed the discovery and development of many potential therapeutics. Here, we describe our efforts to improve yields of landomycins-angucycline family polyketides under investigation as cancer therapeutics-by a genetically modified Streptomyces cyanogenus 136. After simplifying the extraction process from S. cyanogenus cultures, we identified a wavelength at which the major landomycin products are absorbed in culture extracts, which we used to systematically explore culture medium compositions to improve total landomycin titers. Through correlational analysis, we simplified the culture optimization process by identifying an alternative wavelength at which culture supernatants absorb yet is representative of total landomycin titers. Using the subsequently improved sample throughput, we explored landomycin production during the culturing process to further increase landomycin yield and reduce culture time. Testing the antimicrobial activity of the isolated landomycins, we report broad inhibition of Gram-positive bacteria, inhibition of fungi by landomycinone, and broad landomycin resistance by Gram-negative bacteria that is likely mediated by the exclusion of landomycins by the bacterial membrane. Finally, the anticancer activity of the isolated landomycins against A549 lung carcinoma cells agrees with previous reports on other cell lines that glycan chain length correlates with activity. Given the prevalence of natural products produced by Streptomyces, as well as the light-absorbing moieties common to bioactive natural products and their metabolic precursors, our method is relevant to improving the yields of other natural products of interest.

Hematocrit-Independent Sampling Enables White Blood Cell Counts from Patterned Dried Blood Spot Cards.

The accurate and efficient measurement of white blood cell (WBC) counts is vital for monitoring general patient health and can aid in the diagnosis of a range of possible infections or diseases. Even with their importance universally acknowledged, access to WBC counts is largely limited to those with access to phlebotomists and centralized clinical laboratories, which house the instruments that perform the tests. As a result, large populations of people (e.g., those that are home-bound or live in remote locations) lack facile access to testing. Dried blood spot (DBS) cards are often used to bridge these gaps in access to testing by offering the ability to collect blood at home for ambient shipping to laboratories. However, it is well understood that these cards, which are prepared from cellulose cardstocks without further modification, suffer from variabilities in accuracy and precision due to uncontrolled sample spreading and hematocrit effects, which have hindered their use to determine WBC counts. In this paper, we present a method to obtain an accurate WBC count using a patterned dried blood spot (pDBS) card, which comprises collection zones that meter volumes of dried blood. Using an input volume of 75 µL of whole blood, we demonstrate that, unlike the gold standard DBS card (Whatman 903), our pDBS design allows for the collection of replicate zones containing a reproducible, average volume of dried blood (12.1 µL, 7.8% CV) over the range of hematocrits from 25 to 55%. We then used qPCR to quantify the 18S rRNA gene to determine WBC counts from the volumes of blood that are metered in pDBS zones. We observe that WBC counts generated from our method are comparable to those measured with a HemoCue point-of-care WBC analyzer. Our approach to using pDBS cards as a blood collection device has the potential to support at-home sampling and other patient populations that need WBC counts but lack access to clinical facilities.

Rapid spectrophotometric detection for optimized production of landomycins and characterization of their therapeutic potential.

Microbial derived natural products remain a major source of structurally diverse bioactive compounds and chemical scaffolds that have potential as new therapeutics to target drug resistant pathogens and cancers. In particular, genome mining has revealed the vast number of cryptic or low yield biosynthetic gene clusters in the genus Streptomyces . Here, we describe our efforts to improve yields of landomycins - angucycline family polyketides under investigation as cancer therapeutics - by a genetically modified Streptomyces cyanogenus 136. After simplifying the extraction process from S. cyanogenus cultures, we identified a wavelength at which the major landomycin products absorb in culture extracts, which we used to systematically explore culture medium compositions to improve total landomycin titers. Through correlational analysis, we simplified the culture optimization process by identifying an alternative wavelength at which culture supernatants absorb yet is representative of total landomycin titers. Using the subsequently improved sample throughput, we explored landomycin production during the culturing process to further increase landomycin yield and reduce culture time. Testing the antimicrobial activity of the isolated landomycins, we report broad inhibition of Gram-positive bacteria, inhibition of fungi by landomycinone, and broad landomycin resistance by Gram-negative bacteria that is likely mediated by exclusion of landomycins by the bacterial membrane. Finally, the anticancer activity of the isolated landomycins against A549 lung carcinoma cells agrees with previous reports on other cell lines that glycan chain length correlates with activity. Given the prevalence of natural products produced by Streptomyces , as well as the light-absorbing moieties common to bioactive natural products and their metabolic precursors, our method is relevant to improving the yields of other natural products of interest.

Generating signals at converging liquid fronts to create line-format readouts of soluble assay products in three-dimensional paper-based devices.

The correct interpretation of the result from a point-of-care device is crucial for an accurate and rapid diagnosis to guide subsequent treatment. Lateral flow tests (LFTs) use a well-established format that was designed to simplify the user experience. However, the LFT device architecture is inherently limited to detecting analytes that can be captured by molecular recognition. Microfluidic paper-based analytical devices (µPADs), like LFTs, have the potential to be used in diagnostic applications at the point of care. However, µPADs have not gained significant traction outside of academic laboratories, in part, because they have often demonstrated a lack of homogeneous shape or color in signal outputs, which consequently can lead to inaccurate interpretation of results by users. Here, we demonstrate a new class of µPADs that form colorimetric signals at the interfaces of converging liquid fronts (i.e., lines) to control where colorimetric signals are formed without relying on capture techniques. We demonstrate our approach by developing assays for three classes of analytes-an ion, an enzyme, and a small molecule-to measure using iron(III), acetylcholinesterase, and lactate, respectively. Additionally, we show these devices have the potential to support multiplexed assays by generating multiple lines in a common readout zone. These results highlight the ability of this new paper-based device architecture to aid the interpretation of assays that create soluble products by using flow to constrain those colorimetric products in a familiar, line-format output.

Microsampling tools for collecting, processing, and storing blood at the point-of-care.

In the wake of the COVID-19 global pandemic, self-administered microsampling tools have reemerged as an effective means to maintain routine healthcare assessments without inundating hospitals or clinics. Finger-stick collection of blood is easily performed at home, in the workplace, or at the point-of-care, obviating the need for a trained phlebotomist. While the initial collection of blood is facile, the diagnostic or clinical utility of the sample is dependent on how the sample is processed and stored prior to transport to an analytical laboratory. The past decade has seen incredible innovation for the development of new materials and technologies to collect low-volume samples of blood with excellent precision that operate independently of the hematocrit effect. The final application of that blood (i.e., the test to be performed) ultimately dictates the collection and storage approach as certain materials or chemical reagents can render a sample diagnostically useless. Consequently, there is not a single microsampling tool that is capable of addressing every clinical need at this time. In this review, we highlight technologies designed for patient-centric microsampling blood at the point-of-care and discuss their utility for quantitative sampling as a function of collection material and technique. In addition to surveying methods for collecting and storing whole blood, we emphasize the need for direct separation of the cellular and liquid components of blood to produce cell-free plasma to expand clinical utility. Integrating advanced functionality while maintaining simple user operation presents a viable means of revolutionizing self-administered microsampling, establishing new avenues for innovation in materials science, and expanding access to healthcare.

Direct Processing and Storage of Cell-Free Plasma Using Dried Plasma Spot Cards.

Plasma separation cards represent a viable approach for expanding testing capabilities away from clinical settings by generating cell-free plasma with minimal user intervention. These devices typically comprise a basic structure of the plasma separation membrane, unconstrained porous collection pad, and utilize either (i) lateral or (ii) vertical fluidic pathways for separating plasma. Unfortunately, these configurations are highly susceptible to (i) inconsistent sampling volume due to differences in the patient hematocrit or (ii) severe contamination due to leakage of red blood cells or release of hemoglobin (i.e., hemolysis). Herein, we combine the enhanced sampling of our previously reported patterned dried blood spot cards with an assembly of porous separation materials to produce a patterned dried plasma spot card for direct processing and storage of cell-free plasma. Linking both vertical separation and lateral distribution of plasma yields discrete plasma collection zones that are spatially protected from potential contamination due to hemolysis and an inlet zone enriched with blood cells for additional testing. We evaluate the versatility of this card by quantitation of three classes of analytes and techniques including (i) the soluble transferrin receptor by enzyme-linked immunosorbent assay, (ii) potassium by inductively coupled plasma atomic emission spectroscopy, and (iii) 18S rRNA by reverse transcriptase quantitative polymerase chain reaction. We achieve quantitative recovery of each class of analyte with no statistically significant difference between dried and liquid reference samples. We anticipate that this sampling approach can be applied broadly to improve access to critical blood testing in resource-limited settings or at the point-of-care.

Paper-Based Cytometer for the Detection and Enumeration of White Blood Cells According to Their Immunophenotype.

Total and differential white blood cell (WBC) counts are vital metrics used routinely by clinicians to aid in the identification of diseases. However, the equipment necessary to perform WBC counts restricts their operation to centralized laboratories, greatly limiting their accessibility. Established solutions for the development of point-of-care assays, namely lateral flow tests and paper-based microfluidic devices, are inherently limited in their ability to support the detection of WBCs─the pore sizes of materials used to fabricate these devices (e.g., membranes or chromatography papers) do not permit passive WBC transport via wicking. Herein, we identify a material capable of the unimpeded transport of WBCs in both lateral and vertical directions: a coffee filter. Through in situ labeling with an enzyme-labeled affinity reagent, our paper-based cytometer detects WBCs according to their immunophenotype. Using two cultured leukocyte lines (Jurkat D1.1 T cells and MAVER-1 B cells), we demonstrate the specific, colorimetric enumeration of each target cell population across the expected physiological range for total lymphocytes, 1000-4000 cells µL-1. Additionally, we highlight a potential application of this type of device as a screening tool for detecting abnormal cell counts outside the normal physiological range and in subclasses of cell types, which could aid in the identification of certain diseases (e.g., CD4+ T lymphocytes, an important biomarker for HIV disease/AIDS). These results pave the way for a new class of paper-based devices─those capable of controlled white blood cell transport, labeling, capture, and detection─thus expanding the opportunities for low-cost, point-of-care cytometers.

Patterned Dried Blood Spot Cards for the Improved Sampling of Whole Blood.

Dried blood spot (DBS) cards perform many functions for sampling blood that is intended for subsequent laboratory analysis, which include: (i) obviating the need for a phlebotomist by using fingersticks, (ii) enhancing the stability of analytes at ambient or elevated environmental conditions, and (iii) simplifying the transportation of samples without a cold chain. However, a significant drawback of standard DBS cards is the potential for sampling bias due to unrestricted filling caused by the hematocrit of blood, which often limits quantitative or reproducible measurements. Alternative microsampling technologies have minimized or eliminated this bias by restricting blood distribution, but these approaches deviate from clinical protocols and present a barrier to broad adoption. Herein, we describe a patterned dried blood spot (pDBS) card that uses wax barriers to control the flow and restrict the distribution of blood to provide enhanced sampling. These patterned cards reproducibly fill four replicate extraction zones independent of the hematocrit effect. We demonstrate a 3-fold improvement in accuracy for the quantitation of hemoglobin using pDBS cards compared to unpatterned cards. Patterned cards also facilitate the near quantitative recovery (ca. 95%) of sodium with no evidence of a statistically significant difference between dried and liquid blood samples. Similarly, the recovery of select amino acids was conserved in comparison to a recent report with improved intercard precision. We anticipate that this approach presents a viable method for preparing and storing samples of blood in limited resource settings while maintaining current clinical protocols for processing and analyzing dried blood spots.

Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10 min, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 and 80 pM limits of detection in 1× phosphate-buffered saline (mock swab) and saliva matrices spiked with cell-culture-generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way toward the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10-minute, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 pM and 80 pM limits of detection in 1×PBS (mock swab) and saliva matrices spiked with cell-culture generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way towards the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP-KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3-to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3-25 nM) exhibited the lowest limits of detection (ca. µg mL-1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual's immune response can impact the interpretation of assay results.

High-Yielding Separation and Collection of Plasma from Whole Blood Using Passive Filtration.

Lateral flow tests and hand-held analyzers facilitate diagnostic testing in resource limited settings and at the point-of-care. However, many of these devices require sample preparation such as plasma separation to remove cells and isolate the liquid portion of blood. Specifically, the separation of plasma from blood is necessary for routine health assessments such as comprehensive metabolic panels and chronic HIV viral load monitoring. Away from laboratories, this type of processing has been addressed by unconventional, hand-operated centrifuge devices (high volume) or plasma separation membranes (PSM) coupled with lateral flow tests (low volume). Herein, we describe a device that separates and stores plasma from undiluted blood using only passive filtration in less than 10 min. Integrating a PSM with a prefilter and absorbent material yields a 3-fold increase in separation efficiency compared to similar devices using passive filtration. We demonstrate the reproducibility of our device across the physiological range of hematocrits (20-50%) with an average recovered plasma volume of 61.7 ± 2.6 µL. Maximum separation efficiency (53.8%, 65.6 ± 3.9 µL plasma) was achieved for a sample of whole blood (30% hematocrit) in 10 min. We evaluate the purity of our plasma sample by quantitation of hemoglobin and report hemolysis as either minimal (≤5%) or undetectable (≤1%). Specific recovery of human IgG, IFN-γ, and HIV-1 RNA indicate the diagnostic utility of plasma obtained from our device is unchanged compared to plasma obtained via centrifugation. Finally, we demonstrate the use of recovered plasma, applied via "stamping", to successfully conduct a commercial lateral flow immunochromatographic assay for tetanus antibodies. This device platform is capable of producing pure plasma samples from blood to facilitate tests in resource limited settings to improve access to healthcare.

Usability as a guiding principle for the design of paper-based, point-of-care devices - A review.

Due to their portability, versatility for supporting multiple assay formats, and potential for resulting in low-cost assays, paper-based analytical devices (PADs) are an increasingly popular format as a platform for the development of point-of-care tests. However, very few PADs have been translated successfully to their intended environments outside of academic settings. Often overlooked as a factor that inhibits translation, usability is a vital characteristic of any successful point-of-care test. Recent advancements in PAD design have demonstrated improved usability by simplifying various aspects of user operation, including sample collection, sample processing, device operation, detection, and readout/interpretation. Field testing at various stages of device design can offer critical feedback about device usability, especially when it involves the proposed end-user or other stakeholders. By highlighting advances in usability, we aim to encourage thoughtful and rigorous design at the academic prototyping stage to address one outstanding hurdle that limits the number of PADs that make it from the benchtop to the point-of-care.

Dual Sample Preconcentration for Simultaneous Quantification of Metal Ions Using Electrochemical and Colorimetric Assays.

A paper-based method for heating preconcentration (PAD-HP) has been developed for the determination of Pb2+, Cd2+, Fe3+, and Ni2+. The design of our heating system was evaluated for dual quantification of ions using electrochemical and colorimetric methods simultaneously. The PAD-HP was used to detect Pb2+ and Cd2+ by anodic stripping voltammetry and to detect Fe3+ and Ni2+ by colorimetric reactions. Assay conditions were optimized by evaluating performance when changing the concentration of the colorimetric reagent, eluent volume, electrolyte concentration, and electrochemical parameters. Limits of detection (LOD) were determined to be 0.97 and 2.33 µg L-1 for Pb2+ and Cd2+ (via voltammetry) and 0.03 and 0.04 mg L-1 for Fe3+ and Ni2+ (via colorimetric assay), respectively. The relative standard deviations for assays were in the range of 5.76 to 10.12%. We observed that the PAD-HP method significantly enhanced the signal of all metals ions (14-100-fold, depending on the metal) in comparison to paper-based devices that did not use a heating preconcentration system. This PAD-HP method was successfully applied to the determination of metals ions in samples of drinking water, tap water, pond water, and wastewater. These results suggest that our approach can provide a convenient strategy to monitor aqueous samples for heavy metals with high sensitivity and selectivity.

Open software platform for automated analysis of paper-based microfluidic devices.

Development of paper-based microfluidic devices that perform colorimetric measurements requires quantitative image analysis. Because the design geometries of paper-based microfluidic devices are not standardized, conventional methods for performing batch measurements of regularly spaced areas of signal intensity, such as those for well plates, cannot be used to quantify signal from most of these devices. To streamline the device development process, we have developed an open-source program called ColorScan that can automatically recognize and measure signal-containing zones from images of devices, regardless of output zone geometry or spatial arrangement. This program, which measures color intensity with the same accuracy as standard manual approaches, can rapidly process scanned device images, simultaneously measure identified output zones, and effectively manage measurement results to eliminate requirements for time-consuming and user-dependent image processing procedures.

Opportunities in the Synthesis and Design of Radioactive Thin Films and Nanoparticles.

Studies of radioactive isotopes at the liquid-solid or gas-solid interface are enabling a detailed mechanistic understanding of the effects of radioactive decay on physical, biological, and chemical systems. In recent years, there has been a burgeoning interest in using radioactive isotopes for both imaging and therapeutic purposes by attaching them to the surface of colloidal nanoparticles. By merging the field of nanomedicine with the more mature field of internal radiation therapy, researchers are discovering new ways to diagnose and treat cancer. In this Perspective, we discuss state-of-the-art radioactive thin films as applied to both well-defined surfaces and more complex nanoparticles. We highlight the design considerations that are unique to radioactive films, which originate from the damaging and potentially self-destructive emissions produced during radioactive decay, and highlight future opportunities in the largely underexplored area between radioisotope chemistry and nanoscience.

Magnetic Levitation in Chemistry, Materials Science, and Biochemistry.

All matter has density. The recorded uses of density to characterize matter date back to as early as ca. 250 BC, when Archimedes was believed to have solved "The Puzzle of The King's Crown" using density.[1] Today, measurements of density are used to separate and characterize a range of materials (including cells and organisms), and their chemical and/or physical changes in time and space. This Review describes a density-based technique-magnetic levitation (which we call "MagLev" for simplicity)-developed and used to solve problems in the fields of chemistry, materials science, and biochemistry. MagLev has two principal characteristics-simplicity, and applicability to a wide range of materials-that make it useful for a number of applications (for example, characterization of materials, quality control of manufactured plastic parts, self-assembly of objects in 3D, separation of different types of biological cells, and bioanalyses). Its simplicity and breadth of applications also enable its use in low-resource settings (for example-in economically developing regions-in evaluating water/food quality, and in diagnosing disease).

Detection of cardiovascular disease associated miR-29a using paper-based microfluidics and surface enhanced Raman scattering.

The development of viable point-of-care diagnostic formats is integral to achieving better patient care and improved outcomes. The need for robust and low-cost tests is especially important in under-resourced and rural settings. Perhaps the greatest challenge is ensuring that an untrained individual is capable of operating and interpreting the test, out with a care facility. Here we present a paper-based diagnostic device capable of sensing miR-29a using both colorimetric and surface enhanced Raman scattering (SERS) analysis. Rather, than carry out the two types of analyses in tandem, we envisage that the colorimetric output is easy enough to be interpreted by the untrained-individual administering the test to provide them with qualitative feedback. If deemed positive, the test can be further validated at a centralized care facility using a handheld-Raman spectrometer to provide a semi-quantitative result. Detection of miR-29a, a microRNA associated with myocardial infarction, was achieved at a level of pg µL-1 through the combination of three-dimensional paper-based microfluidics, colorimetric detection, and surface enhanced Raman scattering (SERS) analysis. RGB analysis of the colorimetric output generated from samples containing miR-29a at different concentrations (18-360 pg µL-1) showed differentiation from the control sample, however significant repeat variability indicated that it could not be used for quantifying miR-29a levels. However, the SERS analysis exhibited greater reproducibility at varying concentrations, achieving an LoD of 47 pg µL-1. The union of the paper-based device and the two analysis methods resulted in the production of a sensitive, reproducible and facile, point of care test (POCT), which paves the way for future implementation in the diagnosis of a range of diseases.