SERS-based Immunoassay in a Microfluidic System for the Multiplexed Recognition of Interleukins from Blood Plasma: Towards Picogram Detection

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.

Figure 1

Sequential steps for the formation of a SERS-based multiplex immunoassay. (A) The capturing substrate, (B) the mixture of interleukins (IL-6, IL-8, IL-18) in human blood plasma injection, (C) the parallel approach (D) the simultaneous multiplex configuration.

Figure 2

(a,b,c) SERS spectra of FC, p-MBA, and DTNB adsorbed onto AuNPs (Raman reporter-AuNPs), and (a’,b’,c’) SERS spectra of FC, p-MBA, and DTNB–labeled immune AuNPs (antibody-Raman reporter-AuNPs), respectively.

Figure 3

(A) SERS responses in the DA-1 chamber of microfluidic immunoassay during detection of IL-18 in human blood plasma sample according to the subsequent steps of parallel approach: (a) after the mixture of equal amounts of three kinds of Raman reporter-labeled Au nanoparticles was delivered and incubated (5 min) onto the anti-IL18 modified Ag-Au SERS-active surface; (b–f) after subsequent washing by PBS buffer solution. (B) Typical SERS responses for increasing concentration of target IL-6, IL-8, and IL-18 interleukins in blood plasma during a simultaneous multiplexed approach.

Figure 4

(A) SERS spectra obtained for increasing concentration of IL-18: (a) 0.0; (b) 0.005; (c) 0.01; 0.1; (e) 0.5; (f) 1.0; (g) 2.5; (h) 5.0; (i) 10.0; (j) 30.0 ng·ml⁻¹ in blood plasma. (B) The relationship between the intensity of the marker band at 1326 cm⁻¹ versus the concentration of IL-18 in the range from 0 ng·ml⁻¹ to 30 ng·ml⁻¹.

Figure 5

The concentration – intensity calibration curves obtained for simultaneous multiplexed detection of three IL-6, IL-8, and IL-18 interleukins from blood plasma samples. Each SERS spectrum was averaged from 20 measurements at different places across the SERS surface using the mapping mode. The error bars indicate the standard deviations from twenty measurements at different spots for each concentration.