A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer

Abstract

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

Keywords: antibody microarrays; human kallikrein 2; porous silicon; prostate cancer.

Figure 1

Schematic of the P-Si chip immunoassay procedure, starting with the dispensing of hK2 capture antibodies onto the porous silicon surface. The porous silicon chip with physically adsorbed antibodies is placed into an assay well made of polydimethyl-polysiloxane (PDMS), and hK2-containing serum samples are added; the size of the PDMS assay is well suited for each P-Si chip and makes it easy to perform parallel pipetting. Subsequently, detection antibody (polyclonal primary and Alexa 488 labeled secondary antibody) is used for measuring fluorescent signals under a microscope.

Figure 2

Porous silicon (P-Si) matrix used for microarrays. P-Si chips and PDMS wells (a) Capture antibody spotted P-Si chips were located in the wells to start the immunoassay. The scanning electron micrographs show a sequential zoom into a typical surface; (b) Macro-pores of micrometer size are clearly seen, combined with a micro- and nano-morphology (pore size around sub-µm to µm).

Figure 3

Titration series of hK2 in buffer (PBS) solution at three different concentrations of the capturing antibody 6B7 (75 µg/mL, 100 µg/mL and 145 µg/mL). The LOD was found to be 1 pg/mL when the capturing antibody was 75 µg/mL and was reduced to 100 fg/mL when the capturing antibody concentrations were 100 µg/mL. The LOD became 1 pg/mL again when concentration of the antibody was 145 µg/mL.

Figure 4

hK2-spiked human female serum analyzed with the sandwich microarray at two different capturing antibody concentrations (75 µg/mL and 100 µg/mL). The LOD was 10 pg/mL and 1 pg/mL when the concentrations of capture antibody were 75 µg/mL and 100 µg/mL, respectively. Increased assay sensitivity was observed with an elevated concentration of the capturing antibody (6B7). The negative signal also increased at the higher concentrations of the capture antibody.

Figure 5

Immunoassay signals of the diluted serum samples. Serum samples were diluted down to 50 times and the samples were immunoassayed on microarray chips. The concentration of capture antibody was 100 µg/mL. Mean spot intensities and coefficients of variant are presented with spot images in the left panel.

Figure 6

Cross-reaction tests of hK2 antibody spots against PSA spiked serum. Immunoassay signal of negative control (female serum sample) was compared with four PSA-spiked serum samples (5, 50 and 500 ng/mL and 5 µg/mL). HK2 capture antibody was spotted on P-Si chips at a concentration of 100 µg/mL.