Antibody Screening Using a Biophotonic Array Sensor for Immune System Response Profile

The effect of human serum albumin (HSA) on an immunokinetic assay for an antibody to bovine serum albumin has been determined in model serum solutions with HSA concentrations in the range 0 to 450 lM (0–30 mg ml). The assay is performed on two plasmon-based detection platforms: a continuous gold surface and a nanoparticle-based array reader. The assay has a minimum detection concentration of 760 ± 160 pM (120 ± 25 ng ml) in phosphate-buffered saline, falling to 2.5 ± 0.7 nM (380 ± 100 ng ml) in physiological HSA concentration. The concentration of HSA correlates with the refractive index of the solution, and this may be used to calibrate assay response. The addition of the charged chaotrope SCN in 150 mM concentration improves the reproducibility and consistency of the assay, with a minimum detection concentration of 2.9 ± 0.5 nM (440 ± 80 ng ml). The effect of high concentrations of HSA on the immunokinetic assay can be corrected with a measurement of bulk refractive index in a reference channel. Ó 2010 Elsevier Inc. All rights reserved. Measurement of multiple biomarker concentrations in blood serum or whole blood has an obvious benefit over single-biomarker studies, such as for prostate or ovarian cancer [1,2], resulting in consequent advantages for disease diagnosis. Biomarker detection in serum requires low concentrations of a target protein to be measured against the ‘‘background” of large concentrations of other blood proteins [3]. In addition, the concentrations of these background proteins change from patient to patient, with the level of the most abundant protein in blood, human serum albumin (HSA), varying in concentration over the range of 35 to 50 mg ml [4], including healthy and diseased patients. Serum albumin levels are also being investigated as a predictor of disease progression and mortality in conjunction with other serum proteins [5–7]. However, detection of specific biomarkers must still be made against all of the changes in the whole proteome during onset of disease, and in many cases this proves to be rather challenging; for example, more than 10 orders of magnitude separate HSA concentrations from those of cytokine biomarkers [3]. Biomarker concentrations in complex fluids have been monitored, label free, using a number of surface plasmon resonance (SPR) platforms, but these are usually limited to one or two markers in each experiment [8–11]. Several SPR-based imaging technologies have been used to detect multiple biological analytes with array format sensors [12,13], including nanoparticle scattering immunoassay [14–16]. The latter relies on the change in the localized plasmon properties of biofunctionalized nanoparticles on antibody–antigen binding events. The real-time kinetic response is related to the concentration of the biomarker in sample solutions, producing an immunokinetic assay. The nanoparticle localized plasmon field is only sensitive to a change in refractive index (RI) of analyte solution in a small region above the sensor surface. The penetration depth of the particle plasmon field is typically of its radius, which may be compared with the penetration depth of the propagating plasmon on a continuous gold surface of 200 to 300 nm [16,17]. The smaller sensing depth results in better overlap, with an active biolayer that partially compensates for the lower bulk RI sensitivity of the nanoparticles as compared with continuous surface plasmon. This RI contrast confers an advantage on particle-based immunokinetic assays for the detection of antibodies in complex media such as delipidized blood serum [16]. We have been exploiting the nanoparticle plasmon field advantages for complex fluid analysis by developing a nanoparticle Array Reader platform [14–16] where seed nanoparticles are printed in an array format from solution using an inkjet printer. Nanoparticles are grown from the seed particles to larger 130-nm truncated polyhedra to provide bright scatter centers. The array spots are illuminated in a total internal reflection configuration, and the scattered light is monitored in real time. The temporal variation of the immunofunctionalized spots leads to an immunokinetic 0003-2697/$ see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab. 2010.05. 015 * Corresponding author. E-mail address: andrew.m. shaw@exeter.ac. uk (A.M. Shaw). 1 Abbreviations used: HSA, human serum albumin; SPR, surface plasmon resonance; RI, refractive index; ELISA, enzyme-linked immunosorbent assay; PBS, phosphatebuffered saline; RIU, refractive index units; EDC, N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride; NHS, N-hydroxysuccinimide; MHDA, 16-mercaptohexadecanoic acid; BSA, bovine serum albumin; aBSA, antibodies to BSA; IgG, immunoglobulin G; SAM, self-assembled monolayer. Analytical Biochemistry 405 (2010) 114–120