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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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Related in: MedlinePlus

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).
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sensors-15-11972-f002: 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).

Mentions: A 45 mm× 70 mm size and 5 mm thickness polydimethylpolysiloxane (PDMS) slab was prepared to well format as seen in Figure 2. The slab was fabricated with a Syglad 184 kit (Dow Corning Toray, Tokyo, Japan) that contains a PDMS monomer base and a curing elastomer reagent. The base and curing reagent were mixed in 10:1 weight ratio, and poured over a flat surface (i.e., silicon or glass). The mixture was kept at 0.02 MPa for 30 min in a vacuum chamber to remove the trapped air bubbles from the liquid PDMS. After removing the bubbles, the mixture were baked at 70 °C for 1 h. Finally, 9 mm holes were punched on the PDMS slab to set up PDMS wells.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

sensors-15-11972-f002: 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).
Mentions: A 45 mm× 70 mm size and 5 mm thickness polydimethylpolysiloxane (PDMS) slab was prepared to well format as seen in Figure 2. The slab was fabricated with a Syglad 184 kit (Dow Corning Toray, Tokyo, Japan) that contains a PDMS monomer base and a curing elastomer reagent. The base and curing reagent were mixed in 10:1 weight ratio, and poured over a flat surface (i.e., silicon or glass). The mixture was kept at 0.02 MPa for 30 min in a vacuum chamber to remove the trapped air bubbles from the liquid PDMS. After removing the bubbles, the mixture were baked at 70 °C for 1 h. Finally, 9 mm holes were punched on the PDMS slab to set up PDMS wells.

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