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We have developed a disposable point-of-care (POC) aptamer-based biosensor for the detection of salivary cortisol. Nonstressful and noninvasive sampling of saliva compared to that of blood makes saliva an attractive biological matrix in developing POC devices for biomarker monitoring. Aptamers are attractive as recognition elements for multiple reasons, including their specific chemical synthesis, high stability, lack of immunogenicity, and cell-free evolution. A duplex aptamer conjugated to the surface of Au nanoparticles (AuNPs) by Au–S bonds is utilized as the sensor probe in a lateral flow assay (LFA) device. The addition of saliva samples containing cortisol makes the cortisol–aptamer undergo conformational changes and dissociate from the capture probe. Increasing cortisol concentration in the dispensed saliva sample results in increased dissociation and leads to increased binding of AuNP conjugate on the test line. Therefore, the color intensity of the test line on the LFA is a direct function of the concentration of cortisol in saliva. This simple and fast method provides detection in the cortisol range of ∼0.5–15 ng/mL, which is in the clinically accepted range for salivary cortisol. The limit of detection was 0.37 ng/mL, and the accuracy was confirmed by enzyme-linked immunosorbent assay (ELISA) testing results. High selectivity was observed for salivary cortisol against other closely related steroids and stress biomarkers present in saliva.

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Introduction Novel and robust point-of-care (POC) biosensors were designed and developed for the detection of two inherited blood disorders; glucose-6-phosphate dehydrogenase (G6PD) and phenylketonuria (PKU), with PreQuine Systems, Fig. 1a. The G6PD enzyme performs a critical function in human biochemistry. G6PD deficiency is among the most common enzyme pathology that affects 400 million people worldwide and is mainly found in malaria-endemic regions. Malaria affects over 200 million people yearly with 440,000 deaths. Malaria caused by Plasmodium vivax and by Plasmodium oval threatens over 2 billion people globally and sickens tens of millions annually. While treatments such as primaquine and tafenoquine provide a cure, over 8% of the global population are contraindicated due to inherited G6PD deficiency, as these treatments can cause serious, and often life-threatening, anemia. Therefore, a robust, user-friendly, and reliable test is necessary for diagnosing the G6PD deficiency to prevent hemolytic disorders while treating malaria. PreQuine is a novel platform for simultaneous quantification of G6PD and hemoglobin (Hgb) concentrations. PKU is a genetic disorder that causes a build-up of amino acid, specifically L-phenylalanine (Phe), in the blood. PKU is the most common amino acid metabolic disorder and occurs in 1 out of every 8,000 newborns globally. Currently, whole blood is collected in EDTA tubes or spotted onto Dried Blood Spot (DBS) Cards by parents, patients, or caregivers. These samples are sent to laboratories for measurement by tandem mass spectrometry, and results can take days to weeks. This complicated process for monitoring and controlling Phe levels results in non-compliance, a decrease in quality of life, as well as increased healthcare costs for treating complications. Once diagnosed with PKU, Phe concentration levels must be monitored and maintained within acceptable limits between 2 – 6 mg/dL. If left untreated, infants can develop intellectual disabilities or suffer from other common side effects such as seizures, delayed development, behavioral problems, and psychiatric disorders. We have developed a vertical flow colorimetric assay for the quantitative determination of Phe in biological specimens. The assay requires only 15 µL of blood, the Phe within the specimen reacts with the reagent layer to produce an end-point color. This POC platform can be used at home, in the hospital, or at a clinician’s office to measure Phe concentrations and diagnose a patient with PKU. Methods The PreQuine Platform is a novel POC diagnostic test for simultaneous quantification of G6PD and Hgb in malaria patients, Fig.1b. A 10μL finger-stick blood sample is collected via a small capillary tube. The capillary tube is placed directly into a micro-centrifuge tube containing a fixed volume of lysing solution to lyse red blood cells, and a test strip is directly placed into a hand-held meter. The G6PD enzyme and Hgb are liberated into the buffer. Sodium nitrite is immobilized on the transport membrane, which is used to oxidize the hemoglobin. The transport membrane removes cellular debris, and the oxidation of the Hgb is required to eliminate it as a G6PD assay interference. The hemolysate solution migrates laterally over the two detection zones, vertically into the Hgb detection pad and vertically through the optical membrane and into the G6PD detection pad. The optical membrane, above the G6PD detection pad, inhibits the transmittance of light from the LED. The G6PD test strip contains an immobilized substrate, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP+), diaphorase (electron mediator), and a tetrazolium salt indicator. A novel vertical flow assay was also developed for the detection of Phe in blood, Fig. 1c. A 15μL finger-stick blood sample is collected via a small capillary tube and dispensed on the test strip. The test strip is composed of a series of membranes that separate plasma from whole blood. The primary blood separation membrane is composed of glass microfiber with a pore size of 3 – 4 µm, which size excludes 85 to 90 % of cells through immobilized hemagglutinating agents (lectin). The secondary blood separation membrane with a pore size of 2 µm is necessary to remove 100 % cell separation. This membrane also preconditions the plasma to a pH of 7.0 to eliminate interference from tyrosine. The third and final reagent membrane is composed of an anisotropic polysulfone membrane coated with an immobilized tetrazolium salt (WST-5), enzyme (phenylalanine dehydrogenase), electron mediator (diaphorase), and NAD+. Results & Discussion The end-color of the G6PD and Hgb detection pads are measured at 670 nm and 570 nm, with read times of 6 and 1 minute(s), respectively. The PreQuine Platform versus spectrophotometric values revealed an excellent agreement with an R2 = 0.98. Over the analytical range, the average bias was less than 0.75 Unit G6PD per gram of Hgb. The %R was then respectively interpolated to G6PD U/g Hgb to obtain calculated UG6PD/g Hgb. The PKU test results demonstrated exceptional performance over the analytical range of 0-25 mg/dL of Phe (0-6 mg/dL, normal range of Phe in the blood) and showed an excellent correlation with the mass specs values in the range of (0-25 mg/dL) with an R2 = 0.99. The high sensitivity and specificity achieved by the coupling of a diaphorase performs extremely well at low pH needed for the suppression of L-tyrosine interference, along with a highly sensitive tetrazolium salt indicator that acts as a good substrate for the preferred diaphorase. The harmonization of the diaphorase, at a given pH with a specific tetrazolium salt, provided the necessary sensitivity in the analytical range of Phe in blood. Figure 1