Hsin I. Lin

Integrated microfluidic system for rapid screening of CRP aptamers utilizing systematic evolution of ligands by exponential enrichment (SELEX)

The systematic evolution of ligands by exponential enrichment (SELEX) is an experimental procedure that allows screening of given molecular targets by desired binding affinities from an initial random pool of oligonucleotides and oligomers. The final products of SELEX are usually referred as aptamers, which are recognized as promising molecules for a variety of biomedical applications. However, SELEX is an iterative process requiring multiple rounds of extraction and amplification that demands significant time and labor. Therefore, this study presents a novel, automatic, miniature SELEX platform. As a demonstration, the rapid screening of C-reactive protein (CRP) aptamers was performed. By utilizing microfluidic technologies and magnetic beads conjugated with CRP, aptamers with a high affinity to CRP were extracted from a random single-strand deoxyribonucleic acid (ssDNA) pool. These aptamers were further amplified by an on-chip polymerase chain reaction (PCR) process. After five consecutive extraction and amplification cycles, a specific aptamer with the highest affinity was screened automatically. The screened aptamers were used as a recognition molecule for the detection of CRP. The developed microsystem demonstrated fast screening of CRP aptamers and can be used as a powerful tool to select analyte-specific aptamers for biomedical applications.

An integrated microfluidic system for C-reactive protein measurement

This study presents a new microfluidic chip integrated with pneumatic micropumps, normally close microvalves and vortex-type micromixers for C-reactive protein (CRP) measurement. CRP is a protein produced during the inflammation process. It has been reported that CRP in serum can be used for risk assessment of cardiovascular diseases. In this study, CRP measurements were performed by using the integrated microfluidic chip incorporated with magnetic beads. The magnetic beads coated with CRP-specific DNA aptamers were used to recognize, purify and enrich the target CRP. The entire process including sample pre-treatment, and the interaction between the target CRP and anti-CRP antibody was automatically performed on a single chip. The chemiluminescence signal was measured using a luminometer to detect the concentration of CRP afterwards. The entire reaction time is less then 25 min, which is only about 20% of the time required when compared to using traditional bench-top machines (150 min). More importantly, the detection limit has been improved from 0.125 to 0.0125 mg/L with only half the amount of reagent consumption. The development of this microfluidic system is promising for fast, accurate, and sensitive detection of CRP.

An integrated microfluidic system for rapid screening of alpha-fetoprotein-specific aptamers.

The systematic evolution of ligands by exponential enrichment (SELEX) is a screening technique that involves the progressive selection of highly specific ligands via repeated rounds of partition and amplification from a large random pool of nucleic acid sequences. The products of this selection process are called aptamers and are either short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) molecules with a high binding affinity to a large variety of target analytes. However, SELEX is a lengthy, labor-intensive, iterative process requiring multiple rounds of extraction and polymerase chain reaction (PCR) amplification. In order to address these problems, this study presents a new integrated microfluidic system consisting of a magnetic bead-based microfluidic SELEX chip and a competitive assay chip to automate the aptamer screening process. More importantly, the selected ssDNA sequences were confirmed to have a high affinity and specificity to the target molecules, using the developed competitive assay chip. With this approach, an aptamer specific to alpha-fetoprotein (AFP), which is a biomarker for liver cancers, has been successfully selected. The screened aptamer was used as a recognition molecule for AFP and has a linear detection range from 12.5 to 800 ng/mL, which was suitable for rapid clinical applications.

A microfluidic system integrated with optical detection devices for automatic detection of C-reactive protein

C-reactive protein (CRP) is commonly known as a clinical marker, which has been identified as an independent predictor for cardiovascular diseases such as heart attack and stroke. In this study, a new microfluidic system for automatic measurement of CRP is reported. The entire process including sample pre-treatment, incubation between target CRP and anti-CRP antibody, developing process and detection can be automatically performed on a single chip. In addition, chemiluminescent signal was measured by using a home-made luminometer to quantify the concentration of CRP. When compared to previous works, not only can the new chip perform the entire process by integrating a new micro-injector, but a new CRP-specific DNA aptamer was also used for CRP measurement. Experimental data showed that the developed system can automate the entire process within 30 minutes with a detection limit of 0.0125 mgL−1, which was superior to previous works.

Rapid measurement of AFP using AFP-specific aptamer on a microfluidic chip

This study presents a new suction-type microfluidic system capable of rapid measurement of alpha-fetoprotein (AFP) by utilizing magnetic bead-based technologies. Two modules, including a suction-type incubator for the magnetic beads to capture AFP and acridinium ester (AE)-labeled anti-AFP antibodies and a microfluidic control module for sample transportation, were integrated into this microfluidic system. With the incorporation of AFP-specific aptamer-conjugated magnetic beads, the target AFP could be recognized and attracted onto the surface of the magnetic beads from the clinical sample. The entire process including two-step incubation and purification process could be automatically performed within 25 min, which is only about 20% of the time required when using a benchtop machine (for about 130 min). Besides, the total sample and reagent volume consumed is only 105 μL, which is significantly less than that required in a large system (410 μL). More importantly, experimental results showed that clinical human serum samples can also be accurately analyzed. The microfluidic system may be promising for point-of-care applications for AFP detection in the future.

An integrated microfluidic system for rapid screening of alpha-fetoprotein aptamers

Systematic evolution of ligands by exponential enrichment (SELEX) is a screening technique that involves a progressive selection of highly specific ligands by performing repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of the selection are usually called aptamers, which are short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (ssRNA) molecules binding with high affinity to a large variety of target analytes. However, SELEX is an iterative process requiring multiple rounds of extraction and polymerase chain reaction (PCR) amplification that demands significant sampling and process time. In order to address these problems, the study presents a new integrated microfluidic system consisting of an automatic SELEX chip and a competitive assay chip to enhance the aptamer screening efficiency. With this approach, an aptamer specific to alpha-fetoprotein (AFP), which is a biomarker for liver cancer, has been successfully screened.

A Magnetic-Bead Based Microfluidic System for Automatic C-Reactive Protein Detection

C-reactive protein (CRP) has been used as a common indicator during inflammation process. It has been also reported that CRP concentration in serum can be used for risk assessment of cardiovascular diseases. In this study, a new microfluidic system for automatic measurement of CRP is developed. When compared to our previous work, the new chip can perform the entire measurement process by integrating a new micro-injector with other functional microfluidic devices. Experimental data show that the developed system can automate the entire process within 35 minutes with a high sensitivity. The development of the new system may provide a promising platform for automatic measurement of the CRP for point-of-care applications.Copyright