PI: Katsuo Kurabayashi Co-PIs: Pei-Cheng Ku, Edgar Meyhofer
In recent years, biomolecular motors (BMMs) ? highly efficient molecular machines that nature has evolved for over millions of years ? have been employed in miniaturized analysis systems and play important roles in bionanotechnology applications, such as biosensing, molecular sorting, fluidic pumping, micromechanical powering, and molecular assembly. They are compact with a nanometer size, yield robust movement in a fluidic environment, and are readily fueled by adenosine triphosphate (ATP) containing solution. This eliminates the need for an external energy source for micro/nanofluidic actuation. In addition, BMMs efficiently manipulate individual biological molecules and proteins, making possible the development of a motor protein-based biosensing system with a nanoscale mass transport/concentration function. This NSF award by Biosensing/CBET program supports research by Professors Kurabayashi, Ku, and Meyhofer at the University of Michigan on the development of a new biosensing chip technology, namely the biomolecular motor (BMM) smart microarrays, which allows high-throughput, ultrasensitive (at attomolar concentrations) biosensing for multiplexed on-chip protein binding assays. Incorporating a BMM-based mass transport/sensing mechanism in a microfluidic system, the BMM smart microarrays enable autonomous sample handling that involves specific binding, sorting, transporting, and concentrating of multiple target analytes via kinesin motor protein-driven microtubules. The proposed method combines biomolecular motors, photonics and nanofluidics in a single biosensor to simultaneously transport and concentrate large numbers (>10) of molecular analytes to specific detectors for ultra-sensitive quantification. The proposed effort will have broader impact on clinical applications such as stratified medicine and personalized medicine through developing a high-throughput ultra-sensitive multiplexed biomolecular sensing method. The aimed multiplexed biosensing technology will allow for monitoring of the early-stage subtle onset of diseases and early warning of biological threats. The proposed fundamental studies towards combining bionanotechnology and LED-based solid state lighting technology for ultrasensitive multiplexed protein sensing can ultimately be extended to enable the early detection of diseases with a very simple and robust battery-operated handheld module setting. This may open the door for the development of a new commercial product for point-of-care applications under an environment of limited resources. The fundamental knowledge gained from this research will be assimilated the PIs? graduate courses on nanobiomechanics, MEMS, and photoelectronic device technology.
In this project, the involved graduate and undergraduate students will be trained to obtain integrated knowledge and skills in MEMS technology, micro/nano manufacturing, biophysics, biochemistry, and photonics, in collaboration with researchers across several fields. The students? communication and networking skills will grow through their presentations at national/international MEMS and Nanotechnology conferences, and research will be incorporated in the PIs? interdisciplinary graduate courses in MEMS and Nanomanufacturing. Summer interns from underrepresented groups will be actively recruited to this project through the National Nanotechnology Infrastructure Network (NNIN) Research Experience for Undergraduates Program (REU) supported by the NSF.
This research represents transformative potential because it (1) presents the first technique that demonstrates the use of BMM-based nanoscale mass transport and biosensing for multiplexed biosensing; and (2) provides a new approach to realizing robust, cost-effective, simple point-of-care clinical diagnostics with advanced scientific knowledge on controlling BMMs within a man-made engineering structure and on obtaining weak biofluorescent signals at a high signal-to-noise ratio for low-concentration samples.
In recent years, biomolecular motors (BMMs) – highly efficient molecular machines that nature has evolved for over millions of years – have been employed in miniaturized analysis systems and play important roles in bionanotechnology applications, such as biosensing, molecular sorting, fluidic pumping, micromechanical powering, and molecular assembly. They are compact with a nanometer size, yield robust movement in a fluidic environment, and are readily fueled by adenosine triphosphate (ATP) containing solution. This eliminates the need for an external energy source for micro/nanofluidic actuation. In addition, BMMs efficiently manipulate individual biological molecules and proteins, making possible the development of a motor protein-based biosensing system with a nanoscale mass transport/concentration function. This NSF award by Biosensing/CBET program supports research by Professors Kurabayashi, Ku, and Meyhofer at the University of Michigan on the development of a new biosensing chip technology, namely BMM molecular detector. The BMM molecular detector has been shown to allow for high-throughput, ultrasensitive biosensing for highly specific on-chip protein binding assays. Successful implementation of the developed BMM detector chip in clinical applications relies on new advancements in conjugation of microtubule (MT) molecular shuttles with antibodies specifically targeting biomarker proteins of infectious diseases. Therefore, the researchers have made significant effort to establish a protocol that reliably achieves MT-antibody conjugation without compromising the biosensing performance of the chip. The researchers have succeeded conjugation of microtubules with antibodies, such as anti-BSA and anti-IL-6 and found its little adverse effect on the motility/functionality of these microtubules. In particular, IL-6 is a cytokine molecule highly relevant to inflammatory infection human diseases. Using these antibody-conjugated microtubules in the developed molecular concentration device, this study has achieved ultrasensitive biosensing of cytokine molecules in a purified sample. With the device fabrication optimized and the material biocompatibility established, the researchers have demonstrated ATP-fueled self-contained sorting and concentrating of fluorescnetly-labled microtubules. We achieved a 100-fold preconcentration gain for self-aggregated microtubules with a 40-50 minute assay time. Along with the development of the BMM molecular detector device, the researchers have developed a microfluidic technology permitting capillarity-based fully passive immunoassay that involves autonomous reagent/sample handling, washing, and analyte detection on a single microfluidic chip without any external pumps and valves. Their device implemented capillarity-driven immunoassays involving 4 sample and 6 reagent solutions within 30 min by orchestrating the functions of on-chip passive components. Notably, this new immunoassay technique reduced the total number of pipetting processes by ~5 times, as compared to assays on multi-well plates (48 vs. 10). This assay technique allowed the researchers to quantify the concentrations of C-reactive protein (CRP) and suppressor of tumorigenicity 2 (ST2) with a detection limit of 8 and 90 pM, respectively. CRP is used as a biomarker for cardiovascular disease and inflammation, and ST2 is a biomarker for graft-versus-host disease (GVHD) as well as cardiac disease. Coupled with the BMM molecular detector, the fully passive capillarity-driven immunoassay technique should be useful for sophisticated, parallel biochemical microfluidic processing in point-of-care settings under limited resources. The demonstrated biosensing technologies have great potential to yield broader impact on clinical applications such as stratified medicine and personalized medicine through developing a high-throughput ultra-sensitive multiplexed biomolecular sensing method. These technologies may be translated to practical use in monitoring of the early-stage subtle onset of diseases and early warning of biological threats. Combined with LED-based solid-state lighting technology, the demonstrated technologies could ultimately be extended to enable the early detection of diseases with a very simple and robust battery-operated handheld module setting. This may open the door for the development of a new commercial product for point-of-care applications under an environment of limited resources. The fundamental knowledge gained from this research has been assimilated the PIs’ graduate courses on nanobiomechanics, MEMS, and photoelectronic device technology. In this project, the involved graduate and undergraduate students have been trained to obtain integrated knowledge and skills in MEMS technology, micro/nano manufacturing, biophysics, biochemistry, and photonics, in collaboration with researchers across several fields. The students’ communication and networking skills have grown through their presentations at national/international MEMS and Nanotechnology conferences, and research has been incorporated in the PIs’ interdisciplinary graduate courses in MEMS and Nanomanufacturing. Summer interns from underrepresented groups were actively recruited to this project through the National Nanotechnology Infrastructure Network (NNIN) Research Experience for Undergraduates Program (REU) supported by the NSF.
