The ongoing revolution in fundamental biology and clinical discovery critically hinges on the availability of diagnostic tools capable of decentralized point-of-care measurements to provide immediate quantitative information at the bedside or in the clinic. The case of immune monitoring for practical medical treatment, fast, accurate and high throughput analysis of multiple cytokines using a small sample volume is highly required to precisely determine the rapidly changing immune status of patients in different inflammatory disease conditions. The current “gold standard” clinical technology is mainly based on Enzyme-linked immunosorbent assay (ELISA). The complex labelling and washing processes require a total assay time up to more than 72 hours and a sample volume of 0.5- 2 mL per test per patient, which greatly hinders its application for immune monitoring at the point of care. Label-free optical biosensing platforms, where the optical responses are measured at real-time without the need for secondary labelling, offer unique advantages in rapid analysis of complex biological samples. Among these techniques, inclusive of photonic crystal, optical ring resonator, surface plasmon resonance (SPR), fiber optics and interferometry, the nanoplasmonic biosensing based on the localized surface plasmon resonance (LSPR) of noble metal nanoparticles (NPs) has shown exquisite levels of sensing performance. Recent advances in nanomaterials and nanotechnology have spurred the design and fabrication of next generation nanoplasmonic biosensors with various nanostructures, such as nanorod, nano-bipyramid, nano-flower, nano core-shell structure and nanohole arrays. The nanoplasmonic structures offer remarkable potential in sensor sensitivity, tunability, miniaturization, high throughput capability, and large-scale fabrication. The integration of these platforms into highly functional microfluidic devices has provided novel biological interfacing opportunities and promising features for practical biomarker detection. However, the implementation of such devices for real clinical and pharmaceutical settings has still been prohibited due to the deficiency in throughput and manufacturability without necessarily compromising the desirable sensitivity, multiplicity and reliability. Various microarray nanoplasmonic sensing platforms are flourishing owing to the rapid technology evolution in nanofabrication. They hold great promise in massively parallel quantification of multiple analytes by immobilizing specific antibodies in separate spots on a single array. The versatility of parallel detection has significantly increased the throughput of the nanoplasmonic biosensors for multiplex label-free analysis. Yet, majority of these sensing arrays were fabricated using electron beam lithography, direct laser writing, chemical electrodeposition and dip pen nanolithography, which often require dedicated instrumentations, labor intensive fabrication procedures and are extremely costly for large scale production. While there are a few multi-analyte high-throughput nanoplasmonic sensing platforms being developed at a fast pace, it has become clear that the cost, operation complexity, sensing performance, throughput and scalability are equally challenging issues that must be addressed before the technology can be widely accepted in medical practice. Here, we developed a high-throughput, label-free, multiplex LSPR immunoassay based on a facile magnet assisted fabrication method for Fe3O4/Au core-shell nanoparticles (FACSNPs) microarrays. By harnessing the unique superparamagnetic property of the hollow iron oxide nanocore, we demonstrated an easy-to-implement, scalable nanoparticle surface patterning technique for generating regular-shape, well dispersed, individual sensing spots over a large area. Moreover, the strong plasmonic coupling afforded by the decorated gold nanoparticles (AuNPs) on the FACSNPs exhibit superior sensitivity to the local refractive index change upon cytokine binding. Incorporating our previous developed LSPR dark-field imaging technique, the FACSNPs microarray biosensors can conduct 384 of test on four different cytokines for each sample with 16 replicates per cytokine-test. The integration of the FACSNPs microarray sensors into a simple optofluidic device allows real-time, massively parallel detection of multiple cytokines with a low limit of detection to ~ 20 pg/mL using 1 mL of real biological samples. The stability, accuracy and reproducibility of the immunoassay are further confirmed by standard ELISA and validated through successful demonstration of its practical use for functional immunophenotyping of tumor-associated macrophage (TAM) differentiation. Using the physical and chemical properties of the FACSNPs that arise from both the intrinsic properties of constituent nanoparticles and their interparticle interactions, we adopted a magnet assisted self-assembly process to pattern uniform antibody functioned microarray on a glass substrate. Following the microarray patterning, we functionalized the FACSNPs with a panel of four cytokine antibodies using parallel microfluidic channels. The functioned FACSNPs were imaged under SEM showing a thick layer of antibody coating on the NPs. The successful antibody function yielded four physically separated sensing regions with each consisting of large numbers of microarray sensors targeting specific cytokines. This permits the multiplex detection of four cytokines in a massively parallel manner with high statistic accuracy. Figure 1
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Jun 12, 2020
Satyendra K Tiwary + 4
Open Access