| 1. |
- Ahn, Ji-Young, et al.
(författare)
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Sol-Gel Derived Nanoporous Compositions for Entrapping Small Molecules and Their Outlook toward Aptamer Screening
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 84:6, s. 2647-2653
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Tidskriftsartikel (refereegranskat)abstract
- This paper reports for the first time the application of sol-gel microarrays for immobilizing nonsoluble small chemicals (Bisphenol-A; BPA). Also, known problems of sol-gel adhesion to conventional microtiter well plate substrates are circumvented by anchoring the sol-gel microspots to a porous silion surface so-called, PS-SG chips. We confirmed low molecular weight chemical immobilization inside a sol-gel network using fluorescein. BPA and the BPA specific aptamer were utilized as a model pair to verify the affinity specific interaction in the PS-SG selection system. The aptamer interacted specifically with BPA in the sol-gel spots, as shown in microarrays forming the letters "L", "U", "N", and "D". Moreover, the bound aptamer was released by heat, recovered, and verified by gel electrophoresis. The developed PS-SG chip platform will be used for screening aptamers against numerous small molecules such as toxins, metabolites, or pesticide residues.
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| 2. |
- Ji-Young, Ahn, et al.
(författare)
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Aptamer microarray mediated capture and mass spectrometry identification of biomarker in serum samples.
- 2010
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 9:11, s. 5568-5573
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Tidskriftsartikel (refereegranskat)abstract
- Sensitive detection of molecular biomarkers in clinical samples is crucially important in disease diagnostics. This paper reports the developement of an aptamer microarray platform combined with sol-gel technology to identify low-abundance targets in complex serum samples. Because of the nanoporous structure of the sol-gel, a high capacity to immobilize the affinity specific aptamers is accomplished which allows binding and detection of target molecules with high sensitivity. The captured protein is digested in situ and the obtained digest was analyzed by ESI-MS without any interference from the affinity probe. TBP (TATA Box Protein) and its specific aptamers were chosen as a model system. A proof of concept with protein concentrations ranging between nanomolar to micromolar is reported, showing a good linearity up to 400 nM when characterized in an aptamer sandwich assay. Moreover, as low as 0.001% of target protein present in total serum proteins could be identified without any pretreatment step using ESI MS/MS mass spectrometry. We believe this novel strategy could become an efficient method for aptamer-based biomarker detection linked directly to mass spectrometry readout.
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| 3. |
- Lee, SangWook, et al.
(författare)
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A cross-contamination-free SELEX platform for a multi-target selection strategy
- 2013
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Ingår i: Biochip Journal. - : Springer Science and Business Media LLC. - 2092-7843 .- 1976-0280. ; 7:1, s. 38-45
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Tidskriftsartikel (refereegranskat)abstract
- Multi-target aptamer selection, known as multiplex systematic evolution of ligands by exponential enrichment (SELEX), is rapidly drawing interest because of its potential to enable high-speed, high-throughput aptamer selection. The parallelization of chemical processes by integrating microfluidic unit operations is a key strategy for developing a multiplex SELEX process. One of the potential problems with on-chip multiplexing chemical processes is cross-contamination. In order to avoid this, we propose a microfluidic network platform that uses pneumatic valves to allow the serial loading and incubation of aptamers with sol-gel entrapped target proteins. After target binding inside the sol-gels, the cross-contamination-free parallel elution of specifically bound aptamers is performed. The platform allows selective binding with five different targets immobilized in sol-gel spots. When eluting bound species, cross-contamination is avoided by sealing the adjacent elution chambers from each other using the pneumatic microvalves. Consequently, we demonstrate specific aptamer binding to the respective protein target and subsequent aptamer elution without any cross-contamination. This proof of concept opens the way to increased automation and microscale parallel processing of the SELEX methodology.
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| 4. |
- Lee, SangWook, et al.
(författare)
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Improved porous silicon microarray based prostate specific antigen immunoassay by optimized surface density of the capture antibody
- 2013
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Ingår i: Analytica Chimica Acta. - : Elsevier BV. - 1873-4324 .- 0003-2670. ; 796, s. 108-114
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Tidskriftsartikel (refereegranskat)abstract
- Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL(-1), 80 pg mL(1), and 800 fg mL(-1) when arraying the PSA antibody, H117 at the concentration 15 mu g mL(-1), 35 mu g mL(-1), and 154 mu g mL(-1), respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL(-1) to 500 ng mL(-1). The microarray showed a LOD of 800 fg mL(-1) and a dynamic range of 800 fg mL(-1) to 80 ng mL(-1) in serum spiked samples. (C) 2013 Elsevier B.V. All rights reserved.
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| 5. |
- Lee, Sang Wook, et al.
(författare)
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A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.
- 2015
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Ingår i: Sensors. - : MDPI AG. - 1424-8220. ; 15:5, s. 11972-11987
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Tidskriftsartikel (refereegranskat)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 102 ng/mL).
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| 6. |
- Lee, Sang Wook, et al.
(författare)
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A sol-gel integrated dual-readout microarray platform for quantification and identification of prostate-specific antigen
- 2018
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Ingår i: Analytical Sciences. - : Springer Science and Business Media LLC. - 0910-6340 .- 1348-2246. ; 34:3, s. 317-321
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Tidskriftsartikel (refereegranskat)abstract
- Here, we report a sol-gel integrated affinity microarray for on-chip matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) that enables capture and identification of prostate-specific antigen (PSA) in samples. An anti-PSA antibody (H117) was mixed with a sol-gel, and the mixture was spotted onto a porous silicon (pSi) surface without additional surface modifications. The antibody easily penetrates the sol-gel macropore fluidic network structure, making possible high affinities. To assess the capture affinity of the platform, we performed a direct assay using fluorescein isothiocyanate-labeled PSA. Pure PSA was subjected to on-chip MALDI-TOF-MS analysis, yielding three clear mass peptide peaks (m/z = 1272, 1407, and 1872). The sol-gel microarray platform enables dual readout of PSA both fluorometric and MALDI-TOF MS analysis in biological samples. Here we report a useful method for a means for discovery of biomarkers in complex body fluids.
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| 7. |
- Lee, Sang Wook, et al.
(författare)
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Development of a Sol-gel-assisted Reverse-phase Microarray Platform for Simple and Rapid Detection of Prostate-specific Antigen from Serum
- 2018
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Ingår i: Biochip Journal. - : Springer Science and Business Media LLC. - 1976-0280 .- 2092-7843. ; 12:1, s. 69-74
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Tidskriftsartikel (refereegranskat)abstract
- A sol-gel-based reverse-phase microarray was developed with improved sensitivity for prostatespecific antigen (PSA) from serum. The pore-sizecontrolled 3D sol-gel matrix was created with a large surface area to capture target molecules densely. Using the optically active sol-gel nanocomposites, human female serum was spiked with PSA and assessed using the reverse-phase protein microarray. The reverse-phase assay exhibited a limit of detection of 1 pg/mL for PSA and a dynamic range of 103 orders of magnitude. Notably, the platform matched the demand of the immunoassay in a simple and feasible manner. Moreover, the platform shortened the total assay time with an increased accuracy of diagnosis.
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| 8. |
- Lee, Sang Wook, et al.
(författare)
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Porous silicon microarray for simultaneous fluorometric immunoassay of the biomarkers prostate-specific antigen and human glandular kallikrein 2
- 2016
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Ingår i: Microchimica Acta. - : Springer Science and Business Media LLC. - 0026-3672 .- 1436-5073. ; 183:12, s. 3321-3327
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Tidskriftsartikel (refereegranskat)abstract
- The authors have developed a porous silicon (P-Si) based duplex antibody microarray platform for simultaneous quantitation of the biomarkers prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) in serum. Pore size-controlled P-Si surfaces have an extremely enlarged surface area that enables high-density immobilization of fluorescently labeled antibodies by physical adsorption. Automated microarraying of the antibodies provides a fast and reproducible duplex format of antibody arrays on the P-Si chips placed in the wells of a microtiter plate. The assay platform showed a 100 fg·mL−1 limit of detection for both PSA and hK2, and a dynamic range that extends over five orders of magnitude. After optimization of the density of both capture antibodies, neither the PSA nor the hK2 array showed cross-sensitivity to non-target proteins or other plasma proteins. The microarray was evaluated by titration of PSA and hK2, respectively, in the same serum samples. In our perception, this highly sensitive and selective platform holds promise for improved detection of tumor markers in an early diagnostic stage, but also to monitor the recurrence of prostate cancer. [Figure not available: see fulltext.]
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| 9. |
- Park, Jeewoong, et al.
(författare)
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Acousto-microfluidics for screening of ssDNA aptamer
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a new screening method for obtaining a prostate-specific antigen (PSA) binding aptamer based on an acoustofluidic separation (acoustophoreis) technique. Since acoustophoresis provides simultaneous washing and separation in a continuous flow mode, we efficiently obtained a PSA binding aptamer that shows high affinity without any additional washing step, which is necessary in other screening methods. In addition, next-generation sequencing (NGS) was applied to accelerate the identification of the screened ssDNA pool, improving the selecting process of the aptamer candidate based on the frequency ranking of the sequences. After the 8 th round of the acoustophoretic systematic evolution of ligands by exponential enrichment (SELEX) and following sequence analysis with NGS, 7 PSA binding ssDNA aptamer-candidates were obtained and characterized with surface plasmon resonance (SPR) for affinity and specificity. As a result of the new SELEX method with PSA as the model target protein, the best PSA binding aptamer showed specific binding to PSA with a dissociation constant (K d) of 0.7 nM.
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| 10. |
- Tsuyama, Yoshiyuki, et al.
(författare)
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A 50 µm acoustic resonator microchannel enables focusing 100 nm polystyrene beads and sub-micron bioparticles
- 2023
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Ingår i: Sensors and Actuators B: Chemical. - : Elsevier BV. - 0925-4005. ; 376
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Tidskriftsartikel (refereegranskat)abstract
- The manipulation and focusing of submicron/nanometre-scale objects are becoming increasingly important in the fields of biology, chemistry, and materials science. Acoustofluidics provides for the high throughput and precise manipulation of cells and particles with simple equipment. However, application to submicron/nanoparticles is challenging due to the particle volume dependence of the acoustic radiation force and the relatively larger effect of the viscous drag force on small objects. Here, we present an acoustofluidic device that can focus submicron/nanoparticles, bacterial cells, and extracellular vesicles (EVs) using a high-frequency two-dimensional acoustic standing wave. We fabricate 50 µm × 50 µm square cross-section channels as acoustic resonators and generate a two-dimensional acoustic standing wave at an actuation frequency of 14.9 MHz. This configuration enables a strong acoustic radiation force towards the centre of the microchannel and a cross-section-centred single-vortex stream that does not counteract the acoustic radiation force. Using this device, polystyrene beads ranging in diameter from 50 to 500 nm were focused toward the channel centre, where a focused bead stream width of less than 5 µm was achieved at a flow rate of 3 µL min−1 for 100 nm beads and 9 µL min−1 for 200 nm beads. Furthermore, bacterial cells were successfully focused at a flow rate of 40 µL min−1, and the focusing of EVs was also demonstrated. Our acoustofluidic device can become a promising tool for a wide range of biological analyses targeting submicron cells, viruses, and EVs.
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