Proof of Concept for a Portable Platform for Molecular Diagnosis of Tropical Diseases: On-Chip Ready-to-Use Real-Time Quantitative PCR for Detection of Trypanosoma cruzi or Plasmodium spp.

Although molecular diagnostics is well established in clinical laboratories, its full potential has not been extended to field settings. Typically, diagnostic real-time quantitative PCR (qPCR) reagents require temperature-controlled transportation and storage. Furthermore, thermocyclers are bulky and fragile, requiring good infrastructure for optimal operation. These major hurdles strongly limit use of molecular-based tests in low-resource scenarios. Herein, Trypanosoma cruzi or Plasmodium spp. DNA were detected with qPCR using commercial equipment (ABI7500 instrument) and a prototype platform comprising a portable device and a silicon chip, named Q3-Plus. In addition, a ready-to-use reaction format, where all qPCR reagents are stored on plate or on chip, was compared with the traditional freezer-stored format. No significant differences were observed in detecting T. cruzi or Plasmodium spp. DNA between thermocyclers, as well as between reagents' formats, for storage periods of up to 28 days (at 2°C to 8°C or 21°C to 23°C, respectively). When challenged with patients' samples, the Q3-Plus system performed as efficiently as the standard equipment for Plasmodium spp. DNA detection, showing it to be a valuable solution to malaria point-of-care diagnostics. Detection of T. cruzi DNA in chronic patients' samples using the Q3-Plus system yielded approximately 50% efficiency relative to the ABI7500. These results are essential to support future endeavors to bring molecular diagnostics to the point of care, where most needed.

Key Enabling Technologies for Point-of-Care Diagnostics.

A major trend in biomedical engineering is the development of reliable, self-contained point-of-care (POC) devices for diagnostics and in-field assays. The new generation of such platforms increasingly addresses the clinical and environmental needs. Moreover, they are becoming more and more integrated with everyday objects, such as smartphones, and their spread among unskilled common people, has the power to improve the quality of life, both in the developed world and in low-resource settings. The future success of these tools will depend on the integration of the relevant key enabling technologies on an industrial scale (microfluidics with microelectronics, highly sensitive detection methods and low-cost materials for easy-to-use tools). Here, recent advances and perspectives will be reviewed across the large spectrum of their applications.

Q3: A Compact Device for Quick, High Precision qPCR.

An accurate and easy-to-use Q3 system for on-chip quantitative real-time Polymerase Chain Reaction (qPCR) is hereby demonstrated, and described in detail. The qPCR reactions take place inside a single-use Lab-on-a-Chip with multiple wells, each with 5 to 15 µL capacity. The same chip hosts a printed metal heater coupled with a calibrated sensor, for rapid and accurate temperature control inside the reaction mixture. The rest of the system is non-disposable and encased in a 7 × 14 × 8.5 (height) cm plastic shell weighing 300 g. Included in the non-disposable part is a fluorescence read-out system featuring up to four channels and a self-contained control and data storage system, interfacing with an external user-friendly software suite. Hereby, we illustrate the engineering details of the Q3 system and benchmark it with seamlessly ported testing protocols, showing that Q3 equals the performance of standard commercial systems. Overall, to the best of our knowledge, this is one of the most mature general-purpose systems for on-chip qPCR currently available.

Pharmacogenomic Approach to Selecting Antiplatelet Therapy in Patients With Acute Coronary Syndromes: The PHARMCLO Trial.

BACKGROUND: Although clopidogrel is still frequently used in patients with acute coronary syndromes (ACS), its efficacy is hampered by interpatient response variability caused by genetic polymorphisms associated with clopidogrel's metabolism. OBJECTIVES: The goal of this study was to evaluate whether selecting antiplatelet therapy (clopidogrel, prasugrel, or ticagrelor) on the basis of a patient's genetic and clinical characteristics leads to better clinical outcomes compared with the standard of care, which bases the selection on clinical characteristics alone. METHODS: Patients hospitalized for ACS were randomly assigned to standard of care or the pharmacogenomic arm, which included the genotyping of ABCB1, CYP2C19*2, and CYP2C19*17 using an ST Q3 system that provides data within 70 min at each patient's bedside. The patients were followed up for 12 ± 1 month for the primary composite endpoint of cardiovascular death and the first occurrence of nonfatal myocardial infarction, nonfatal stroke, and major bleeding defined according to Bleeding Academic Research Consortium type 3 to 5 criteria. RESULTS: After enrolling 888 patients, the study was prematurely stopped. Clopidogrel was used more frequently in the standard-of-care arm (50.7% vs. 43.3%), ticagrelor in the pharmacogenomic arm (42.6% vs. 32.7%; p = 0.02), and prasugrel was equally used in both arms. The primary endpoint occurred in 71 patients (15.9%) in the pharmacogenomic arm and in 114 (25.9%) in the standard-of-care arm (hazard ratio: 0.58; 95% confidence interval: 0.43 to 0.78; p < 0.001). CONCLUSIONS: A personalized approach to selecting antiplatelet therapy for patients with ACS may reduce ischemic and bleeding events. (Pharmacogenetics of Clopidogrel in Patients With Acute Coronary Syndromes [PHARMCLO]; NCT03347435).

Rapid and portable, lab-on-chip, point-of-care genotyping for evaluating clopidogrel metabolism.

BACKGROUND: Dual antiplatelet therapy with aspirin and a platelet P2Y12 receptor inhibitors (clopidogrel, prasugrel, ticagrelor) is a cornerstone of antithrombotic treatment in patients with acute coronary syndromes (ACS). Clopidogrel has been the standard of care for nearly a decade; however, its clinical efficacy is influenced by a considerable inter-patient variability in response, clearly associated to cytochrome P (CYP) enzyme genetic variations. We used a novel point-of-care lab-on-chip instrument to genotype ACS patients in order to identify carriers of the ATB-binding cassette ABCB1 3435, CYP2C19*2 and CYPC2C19*17 alleles and adjust the pharmacological approach accordingly. METHODS AND RESULTS: Between October 2012 and January 2013, 160 ACS patients were enrolled at the Cardiology Unit of the Ospedale Niguarda Cà Granda and genotyped at the patients' point-of-care using the newly developed Q3 portable real-time PCR instrument, which remarkably scored the CYP2C19*2, CYP2C19*17, and ABCB1 3435 alleles in a time of 70 min from DNA extraction to final genotype calls; concordance with the other gold-standard genotyping techniques was 100%. CONCLUSIONS: The Q3 instrument proved to be as reliable as the current conventional techniques. As genotyping in the ACS setting cannot be delegated to centralised clinical laboratories for reasons of time, genotyping at the patients' bedside provides an opportunity to conduct large-scale randomised trials in order to assess whether adding genotype data to clinical variables improves clinical outcomes.

Label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance.

In order to improve the efficacy and safety of treatments, drug dosage needs to be adjusted to the actual needs of each patient in a truly personalized medicine approach. Key for widespread dosage adjustment is the availability of point-of-care devices able to measure plasma drug concentration in a simple, automated, and cost-effective fashion. In the present work, we introduce and test a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup, comprised of off-the-shelf components and coupled with DNA-based aptamers specific to the antibiotic tobramycin (467 Da). The core of the T-LSPR setup are aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor. The gold NIs exhibit localized plasmon resonance in the visible range matching the sensitivity of the complementary metal oxide semiconductor (CMOS) image sensor employed as a light detector. The combination of gold NIs on the FTO substrate, causing NIs size and pattern irregularity, might reduce the overall sensitivity but confers extremely high stability in high-ionic solutions, allowing it to withstand numerous regeneration cycles without sensing losses. With this rather simple T-LSPR setup, we show real-time label-free detection of tobramycin in buffer, measuring concentrations down to 0.5 µM. We determined an affinity constant of the aptamer-tobramycin pair consistent with the value obtained using a commercial propagating-wave based SPR. Moreover, our label-free system can detect tobramycin in filtered undiluted blood serum, measuring concentrations down to 10 µM with a theoretical detection limit of 3.4 µM. While the association signal of tobramycin onto the aptamer is masked by the serum injection, the quantification of the captured tobramycin is possible during the dissociation phase and leads to a linear calibration curve for the concentrations over the tested range (10-80 µM). The plasmon shift following surface binding is calculated in terms of both plasmon peak location and hue, with the latter allowing faster data elaboration and real-time display of the results. The presented T-LSPR system shows for the first time label-free direct detection and quantification of a small molecule in the complex matrix of filtered undiluted blood serum. Its uncomplicated construction and compact size, together with the remarkable performances, represent a leap forward toward effective point-of-care devices for therapeutic drug concentration monitoring.

Point-of-care systems for rapid DNA quantification in oncology.

The development of new powerful applications and the improvement in fabrication techniques are promising an explosive growth in lab-on-chip use in the upcoming future. As the demand reaches significant levels, the semiconductor industry may enter in the field, bringing its capability to produce complex devices in large volumes, high quality and low cost. The lab-on-chip concept, when applied to medicine, leads to the point-of-care concept, where simple, compact and cheap instruments allow diagnostic assays to be performed quickly by untrained personnel directly at the patient's side. In this paper, some practical and economical considerations are made to support the advantages of point-of-care testing. A series of promising technologies developed by STMicroelectronics on lab-on-chips is also presented, mature enough to enter in the common medical practice. The possible use of these techniques for cancer research, diagnosis and treatment are illustrated together with the benefits offered by their implementation in point-of-care testing.