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

Lee SW, Hosokawa K, Kim S, Jeong OC, Lilja H, Laurell T, Maeda M - Sensors (Basel) (2015)

Bottom Line: The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization.In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL.The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL).

View Article: PubMed Central - PubMed

Affiliation: Bioengineering Laboratory, RIKEN, Saitama 3510198, Japan. SangWook_L@chem.s.u-tokyo.ac.jp.

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).

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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.
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sensors-15-11972-f006: 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.

Mentions: Since human kallikrein 2 (hK2) has a high homology to prostate specific antigen (PSA) there is a critical need to carefully determine the hK2 assay design against PSA. We therefore performed serum- spiked PSA assays using the hK2 antibody microarrays to investigate the ratio of cross-reaction between PSA and hK2. We dispensed hK2 capture antibody with 100 µg/mL concentration on P-Si substrates and performed immunoassays of PSA-spiked serum at four different concentration (5, 50 and 500 ng/mL and 5 µg/mL). Figure 6 shows the readout microarray signals of PSA-spiked serum with negative control. All four signals of PSA-spiked serum are not significantly different from that of the negative control, from which it can be determined that there is no cross-reaction between hK2 capture antibody and PSA. The developed P-Si microarray platform therefore, is sufficient to quantify of hK2 abundance in serum without worrying about any cross-reaction with PSA.


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

Lee SW, Hosokawa K, Kim S, Jeong OC, Lilja H, Laurell T, Maeda M - Sensors (Basel) (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481930&req=5

sensors-15-11972-f006: 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.
Mentions: Since human kallikrein 2 (hK2) has a high homology to prostate specific antigen (PSA) there is a critical need to carefully determine the hK2 assay design against PSA. We therefore performed serum- spiked PSA assays using the hK2 antibody microarrays to investigate the ratio of cross-reaction between PSA and hK2. We dispensed hK2 capture antibody with 100 µg/mL concentration on P-Si substrates and performed immunoassays of PSA-spiked serum at four different concentration (5, 50 and 500 ng/mL and 5 µg/mL). Figure 6 shows the readout microarray signals of PSA-spiked serum with negative control. All four signals of PSA-spiked serum are not significantly different from that of the negative control, from which it can be determined that there is no cross-reaction between hK2 capture antibody and PSA. The developed P-Si microarray platform therefore, is sufficient to quantify of hK2 abundance in serum without worrying about any cross-reaction with PSA.

Bottom Line: The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization.In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL.The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL).

View Article: PubMed Central - PubMed

Affiliation: Bioengineering Laboratory, RIKEN, Saitama 3510198, Japan. SangWook_L@chem.s.u-tokyo.ac.jp.

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).

Show MeSH
Related in: MedlinePlus