A label-free immunoassay for Flavivirus detection by the Reflective Phantom Interface technology.

Flaviviruses are widespread and cause clinically relevant arboviral diseases that impact locally and as imported travel-related infections. Direct detection of viraemia is limited, being typically undetectable at onset of symptoms. Therefore, diagnosis is primarily based on serology, which is complicated by high cross-reactivity across different species. The overlapping geographical distribution of the vectors in areas with a weak healthcare system, the increase of international travel and the similarity of symptoms highlight the need for rapid and reliable multi-parametric diagnostic tests in point-of-care formats. To this end we developed a bi-parametric serological microarray using recombinant NS1 proteins from Tick-borne encephalitis virus and West Nile virus coupled to a low-cost, label-free detection device based on the Reflective Phantom Interface (RPI) principle. Specific sequential detection of antibodies in solution demonstrates the feasibility of the approach for the surveillance and diagnosis of Flaviviruses.

Multispot, label-free biodetection at a phantom plastic-water interface.

Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, low-cost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.

Multi-spot, label-free immunoassay on reflectionless glass.

Biosensing platforms that combine high sensitivity, operational simplicity and affordable costs find wide application in many fields, including human diagnostics, food and environmental monitoring. In this work, we introduce a label-free biosensing chip made of glass with a single anti-reflective layer of SiO2. This common and economic material coated by a multi-functional copolymer based on dimethylacrylamide enables the detection even in turbid media. The copolymer coating provides covalent immobilization of antibodies onto the surface and prevents the non-specific adsorption of analytes and matrix constituents. The specific capture of target compounds yields a local increase of surface reflectivity measured by a simple imaging system. Chip design and quantitative interpretation of the data are based on a theoretical optical model. This approach enables the multiplex detection of biomolecular interactions with state-of-the-art sensitivity and minimal instrumental complexity. The detection performance is demonstrated by characterizing the interaction between human growth hormone in solution and the corresponding antibodies immobilized on the sensing surface, both in buffer and human serum, obtaining a clear signal for concentrations as small as 2.8 ng/ml.

A fast and simple label-free immunoassay based on a smartphone.

Despite the continuous advancements in bio-molecular detection and fluidic systems integration, the realization of portable and high performance devices for diagnostic applications still presents major difficulties, mostly because of the need to combine adequate sensitivity with low cost of production and operational simplicity and speed. In this context, we propose a compact device composed of a smartphone and a custom-designed cradle, containing only a disposable sensing cartridge, a tiny magnetic stirrer and a few passive optical components. The detection principle is the previously proposed Reflective Phantom Interface that is based on measuring the intensity of light reflected by the surface of an amorphous fluoropolymer substrate, which has a refractive index very close to that of the aqueous sample solution and hosts various antibodies immobilized within spots. The reflectivity of dozens of spots is monitored in real time by the phone׳s camera using the embedded flash LED as the illumination source. We test the performance of the combined device targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV. Target concentrations as low as a few ng/ml can be rapidly and robustly determined by comparing the rate of increase of the signal after the addition of the sample with that measured after the subsequent addition of a standard solution with known concentration. The features of the proposed system enable the realization of novel handheld biosensing devices suitable for those applications where multiple targets have to be rapidly detected even without the presence of trained personnel.