Efficient isolation of encoded microparticles in a degassed micromold for highly sensitive and multiplex immunoassay with signal amplification

Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient p...

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Published in:	Biosensors & bioelectronics Vol. 261; p. 116465
Main Authors:	Jang, Wookyoung, Song, E Loomee, Mun, Seok Joon, Bong, Ki Wan
Format:	Journal Article
Language:	English
Published:	England Elsevier B.V 01.10.2024
Subjects:	air biomarkers Biomarkers - urine Biosensing Techniques - methods biosensors Chorionic Gonadotropin, beta Subunit, Human - analysis Chorionic Gonadotropin, beta Subunit, Human - blood Chorionic Gonadotropin, beta Subunit, Human - isolation & purification Chorionic Gonadotropin, beta Subunit, Human - urine detection limit Dimethylpolysiloxanes - chemistry early diagnosis Equipment Design Female gonadotropins Humans Hydrogel Immunoassay Immunoassay - methods immunoassays isolation techniques Limit of Detection microarray technology Multiplex detection nanopores Particle isolation peroxidase phosphates polydimethylsiloxane precision medicine Pregnancy Protein proteomics Signal amplification urine Vascular Endothelial Growth Factor A - analysis Vascular Endothelial Growth Factor A - isolation & purification Vascular Endothelial Growth Factor A - urine vascular endothelial growth factors Immunoassay Particle isolation Hydrogel Multiplex detection Protein Signal amplification
ISSN:	0956-5663, 1873-4235, 1873-4235
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Abstract	Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields.
AbstractList	Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields. Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields.Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields. Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields.
ArticleNumber	116465
Author	Song, E Loomee Mun, Seok Joon Jang, Wookyoung Bong, Ki Wan
Author_xml	– sequence: 1 givenname: Wookyoung orcidid: 0000-0003-1889-1131 surname: Jang fullname: Jang, Wookyoung – sequence: 2 givenname: E Loomee orcidid: 0009-0000-4414-7694 surname: Song fullname: Song, E Loomee – sequence: 3 givenname: Seok Joon surname: Mun fullname: Mun, Seok Joon – sequence: 4 givenname: Ki Wan surname: Bong fullname: Bong, Ki Wan email: bong98@korea.ac.kr
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Keywords	Immunoassay Particle isolation Hydrogel Multiplex detection Protein Signal amplification
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Snippet	Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision...
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SubjectTerms	air biomarkers Biomarkers - urine Biosensing Techniques - methods biosensors Chorionic Gonadotropin, beta Subunit, Human - analysis Chorionic Gonadotropin, beta Subunit, Human - blood Chorionic Gonadotropin, beta Subunit, Human - isolation & purification Chorionic Gonadotropin, beta Subunit, Human - urine detection limit Dimethylpolysiloxanes - chemistry early diagnosis Equipment Design Female gonadotropins Humans Hydrogel Immunoassay Immunoassay - methods immunoassays isolation techniques Limit of Detection microarray technology Multiplex detection nanopores Particle isolation peroxidase phosphates polydimethylsiloxane precision medicine Pregnancy Protein proteomics Signal amplification urine Vascular Endothelial Growth Factor A - analysis Vascular Endothelial Growth Factor A - isolation & purification Vascular Endothelial Growth Factor A - urine vascular endothelial growth factors
Title	Efficient isolation of encoded microparticles in a degassed micromold for highly sensitive and multiplex immunoassay with signal amplification
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