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Abstract
SERS-active nanostructures incorporated into a microfluidic device have been developed for rapid and multiplex monitoring of selected Type 1 cytokine (interleukins: IL-6, IL-8, IL-18) levels in blood plasma. Multiple analyses have been performed by using nanoparticles, each coated with different Raman reporter molecules: 5,5′-dithio-bis(2-nitro-benzoic acid) (DTNB), fuchsin (FC), and p-mercatpobenzoic acid (p-MBA) and with specific antibodies. The multivariate statistical method, principal component analysis (PCA), was applied for segregation of three different antigen-antibody complexes encoded by three Raman reporters (FC, p-MBA, and DTNB) during simultaneous multiplexed detection approach. To the best of our knowledge, we have also presented, for the first time, a possibility for multiplexed quantification of three interleukins: IL-6, IL-8, and IL-18 in blood plasma samples using SERS technique. Our method improves the detection limit in comparison to standard ELISA methods. The low detection limits were estimated to be 2.3 pg·ml−1, 6.5 pg·ml−1, and 4.2 pg·ml−1 in a parallel approach, and 3.8 pg·ml−1, 7.5 pg·ml−1, and 5.2 pg·ml−1 in a simultaneous multiplexed method for IL-6, IL-8, and IL-18, respectively. This demonstrated the sensitivity and reproducibility desirable for analytical examinations.
Highlights
Up to now several analytical procedures have been applied for evaluating cytokine levels in body fluids
The first layer of this sandwich structure is composed of immobilized antibodies against IL-6, IL-8, and IL-18 interleukins captured on a Ag-Au bimetallic SERSactive surface via 6-amino-1-hexanethiol (AHT) layers
The surface-enhanced Raman scattering (SERS)-active substrate used in this study exhibits, besides a uniformly high enhancement factor, high reproducibility, plus the stability of recorded signals across a single substrate and between different substrates
Summary
Up to now several analytical procedures have been applied for evaluating cytokine levels in body fluids. Yokoe et al have measured serum IL-8 levels in 12 heavily pretreated patients with recurrent breast cancer and reported a small increase of IL-8 in those patients with refractory progressive disease and almost no decrease in those with partial response or no change after systemic therapy[9]. Only a few sensors for these immune markers have been proposed in the literature and until now there are no methods which allow the simultaneous quantification and multiplex analysis of interleukins in the body fluids. The development of such methods is extremely important. The SERS technology can be used potentially for the quantitative measurement of analytes with ultrahigh sensitivity and offers nondestructive, reliable, and fast detection of samples, which leads to various practical applications in studying, e.g. nucleic acids and proteins[13], therapeutic agents[14], drugs and trace materials[15], and bacteria cells[16, 17]
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