Reusable and highly sensitive SERS immunoassay utilizing gold nanostars and a cellulose hydrogel-based platform.

The development of robust and sensitive point-of-care testing platforms is necessary to improve patient care and outcomes. Surface-enhanced Raman scattering (SERS)-based immunosensors are especially suited for this purpose. Here, we present a highly sensitive and selective SERS immunoassay, demonstrating for example the detection of horseradish peroxidase (HRP), in a sandwich format. The strength of our biosensor lies in merging: (i) SERS-immunotags based on gold nanostars, allowing exceptional intense SERS from attached Raman probes, covalent attachment of anti-HRP antibodies by a simple chemical method providing exceptional antigen binding activity; (ii) the ease of preparation of the capture platform from a regenerated cellulose-based hydrogel, a transparent material, ideal for microfluidics applications, with low background fluorescence and Raman signal, particularly suited for preserving high activity of the covalently bound anti-HRP antibodies. The sandwich complexes formed were characterised by atomic force microscopy, and by scanning electron microscopy coupled with electron diffraction spectroscopy; and (iii) the robustness of the simple Classical Least Squares method for SERS data analysis, resulting in superior discrimination of SERS signals from the background and much better data fitting, compared to the commonly used peak integral method. Our SERS immunoassay greatly improves the detection limits of traditional enzyme-linked immunosorbent assay approaches, and its performance is better or comparable to those of existing SERS-based immunosensors. Our approach successfully overcomes the main challenges of application at point-of-care, including increasing reproducibility, sensitivity, and specificity, associated with an environmentally friendly and robust design. Also, the proposed design withstands several cycles of regeneration, a feature absent in paper-SERS immunoassays and this opens the way for sensitive multiplexing applications on a microfluidic platform.

Development of a Gold Nanoparticle-Based Lateral-Flow Immunoassay for Pneumocystis Pneumonia Serological Diagnosis at Point-of-Care.

Pneumocystis jirovecii pneumonia (PcP) is a major human immunodeficiency virus (HIV)-related illness, rising among immunocompromised non-HIV patients and in developing countries. Presently, the diagnosis requires respiratory specimens obtained through invasive and costly techniques that are difficult to perform in all patients or implement in all economic settings. Therefore, the development of a faster, cost-effective, non-invasive and field-friendly test to diagnose PcP would be a significant advance. In this study, recombinant synthetic antigens (RSA) of P. jirovecii's major surface glycoprotein (Msg) and kexin-like serine protease (Kex1) were produced and purified. These RSA were applied as antigenic tools in immunoenzymatic assays for detection of specific anti-P. jirovecii antibodies (IgG and IgM) in sera of patients with (n = 48) and without (n = 28) PcP. Results showed that only IgM anti-P. jirovecii levels were significantly increased in patients with PcP compared with patients without P. jirovecii infection (p ≤ 0.001 with both RSA). Thus, two strip lateral flow immunoassays (LFIA), based on the detection of specific IgM anti-P. jirovecii antibodies in human sera samples, were developed using the innovative association of P. jirovecii's RSA with spherical gold nanoparticles (AuNPs). For that, alkanethiol-functionalized spherical AuNPs with ca. ~40 nm in diameter were synthetized and conjugated with the two RSA (Msg or Kex1) produced. These AuNP-RSA conjugates were characterized by agarose gel electrophoresis (AGE) and optimized to improve their ability to interact specifically with serum IgM anti-P. jirovecii antibodies. Finally, two LFIA prototypes were developed and tested with pools of sera from patients with (positive sample) and without (negative sample) PcP. Both LFIA had the expected performance, namely, the presence of a test and control red colored lines with the positive sample, and only a control red colored line with the negative sample. These results provide valuable insights into the possibility of PcP serodiagnosis at point-of-care. The optimization, validation and implementation of this strip-based approach may help to reduce the high cost of medical diagnosis and subsequent treatment of PcP both in industrialized and low-income regions, helping to manage the disease all around the world.