Ruba Khnouf

Cell-Free Protein Synthesis in Microfluidic Array Devices

We report the development of a microfluidic array device for continuous‐exchange, cell‐free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high‐throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl‐transferase, and luciferase. Protein expression in the device lasts 5–10 times longer and the production yield is 13–22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield.

Cell-Free Expression of Soluble and Membrane Proteins in an Array Device for Drug Screening

Enzymes and membrane protein receptors represent almost three-quarters of all current drug targets. As a result, it would be beneficial to have a platform to produce them in a high-throughput format for drug screening. We have developed a miniaturized fluid array device for cell-free protein synthesis, and the device was exploited to produce both soluble and membrane proteins. Two membrane-associated proteins, bacteriorhodopsin and ApoA lipoprotein, were coexpressed in an expression medium in the presence of lipids. Simultaneous expression of ApoA lipoprotein enhanced the solubility of bacteriorhodopsin and would facilitate functional studies. In addition, the device was employed to produce two enzymes, luciferase and beta-lactamase, both of which were demonstrated to be compatible with enzyme inhibition assays. Beta-lactamase, a drug target associated with antibiotic resistance, was further used to show the capability of the device for drug screening. Beta-lactamase was synthesized in the 96 units of the device and then assayed by a range of concentrations of four mock drug compounds without harvesting and purification. The inhibitory effects of these compounds on beta-lactamase were measured in a parallel format, and the degree in their drug effectiveness agreed well with the data in the literature. This work demonstrated the feasibility of the use of the fluid array device and cell-free protein expression for drug screening, with advantages in less reagent consumption, shorter analysis time, and higher throughput.

Miniaturized fluid array for high-throughput protein expression

We describe a miniaturized fluid array device for high‐throughput cell‐free protein synthesis (CFPS), aiming to match the throughput and scale of gene discovery. Current practice of using E. coli cells for production of recombinant proteins is difficult and cost‐prohibitive to implement in a high‐throughput format. As more and more new genes are being identified, there is a considerable need to have high‐throughput methods to produce a large number of proteins for studying structures and functions of the corresponding genes. The device consists of 96 units and each unit is for expression of one protein; thus up to 96 proteins can be produced simultaneously. The function of the fluid array was demonstrated by expression of a variety of proteins, with more than two orders of magnitude reduction in reagent consumption compared with a commercially available CFPS instrument. The protein expression yield in the device was up to 87 times higher for β‐glucoronidase than that in a conventional microplate. The concentration of β‐galactosidase expressed in the device was determined at 5.5 μg/μL. The feasibility of using the device for drug screening was demonstrated by measuring the inhibitory effects of mock drug compounds on synthesized β‐lactamase without the need for harvesting proteins, which enabled us to reduce the analysis time from days to hours.

Fabrication optimization of a miniaturized array device for cell-free protein synthesis.

Cell‐free protein synthesis (CFPS) is an attractive alternative to cell‐based protein expression systems because of its advantages including speed, simplicity, and adaptability to various formats. However, two major obstacles exist that have been preventing it from being widely used. One is high cost and the other is low protein synthesis yield. We report here a miniaturized CFPS device that addresses these challenges. The cost saving was achieved by miniaturization, which reduced the reagent consumption by two orders of magnitude. The protein synthesis yield was enhanced by prolonging CFPS reactions through continuous supply of reactants (e.g. nutrients and energy components). The reactants were contained in a feeding solution, which was replenished through a nanoporous membrane and microchannel. The design of the miniaturized device was optimized by running continuous‐exchange CFPS in devices with a variation in the type of membrane, the size of the exchange interface, and the volume ratio of the reaction solution to the feeding solution. The effects of these design variations on the protein synthesis yield have been studied. Furthermore, the design was expanded into a 96‐unit device that can produce a large number of proteins simultaneously, enabling high‐throughput proteomics applications.

Parkinson's disease hand tremor detection system for mobile application.

Abstract Parkinson’s disease currently affects millions of people worldwide and is steadily increasing. Many symptoms are associated with this disease, including rest tremor, bradykinesia, stiffness or rigidity of the extremities and postural instability. No cure is currently available for Parkinson’s disease patients; instead most medications are for treatment of symptoms. This treatment depends on the quantification of these symptoms such as hand tremor. This work proposes a new system for mobile phone applications. The system is based on measuring the acceleration from the Parkinson’s disease patient’s hand using a mobile cell phone accelerometer. Recordings from 21 Parkinson’s disease patients and 21 healthy subjects were used. These recordings were analysed using a two level wavelet packet analysis and features were extracted forming a feature vector of 12 elements. The features extracted from the 42 subjects were classified using a neural networks classifier. The results obtained showed an accuracy of 95% and a Kappa coefficient of 90%. These results indicate that a cell phone accelerometer can accurately detect and record rest tremor in Parkinson’s disease patients.

Protein immobilization on the surface of polydimethylsiloxane and polymethyl methacrylate microfluidic devices

PDMS and PMMA are two of the most used polymers in the fabrication of lab‐on‐chip or microfluidic devices. In order to use these polymers in biological applications, it is sometimes essential to be able to bind biomolecules such as proteins and DNA to the surface of these materials. In this work, we have evaluated a number of processes that have been developed to bind protein to PDMS surfaces which include passive adsorption, passive adsorption with glutaraldehyde cross‐linking, (3‐aminopropyl) triethoxysilane functionalization followed by glutaraldehyde or 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride cross‐linkers. It has been shown that the latter technique—using 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride—results in more than twice the bonding of protein to the surface of PDMS microchannels than proteins binding passively. We have also evaluated a few techniques that have been tested for the functionalization of PMMA microchannels where we have found that the use of polyethyleneimine (PEI) has led to the strongest protein‐PMMA microchannel bond. We finally demonstrated the effect of PDMS curing methodology on protein adsorption to its surface, and showed that increased curing time is the factor that reduces passive adsorption the most.

Cell-Free Protein Synthesis in Microfluidic 96-Well Plates

Cell-free protein synthesis (CFPS) enables rapid protein expression for the structural and functional characterization of proteins. Implementation of CFPS in a microfluidic platform has additional benefits such as reduced reaction volumes and simultaneous expression of multiple proteins. Here, we describe a microfluidic device that is composed of 96 continuous-exchange cell-free protein expression units and produces a protein synthesis yield up to 87 times higher than a conventional batch system.

Improvement of medical content in the curriculum of biomedical engineering based on assessment of students outcomes

BackgroundImprovement of medical content in Biomedical Engineering curricula based on a qualitative assessment process or on a comparison with another high-standard program has been approached by a number of studies. However, the quantitative assessment tools have not been emphasized. The quantitative assessment tools can be more accurate and robust in cases of challenging multidisciplinary fields like that of Biomedical Engineering which includes biomedicine elements mixed with technology aspects. The major limitations of the previous research are the high dependence on surveys or pure qualitative approaches as well as the absence of strong focus on medical outcomes without implicit confusion with the technical ones. The proposed work presents the development and evaluation of an accurate/robust quantitative approach to the improvement of the medical content in the challenging multidisciplinary BME curriculum.MethodsThe work presents quantitative assessment tools and subsequent improvement of curriculum medical content applied, as example for explanation, to the ABET (Accreditation Board for Engineering and Technology, USA) accredited biomedical engineering BME department at Jordan University of Science and Technology. The quantitative results of assessment of curriculum/course, capstone, exit exam, course assessment by student (CAS) as well as of surveys filled by alumni, seniors, employers and training supervisors were, first, mapped to the expected students’ outcomes related to the medical field (SOsM). The collected data were then analyzed and discussed to find curriculum weakness points by tracking shortcomings in every outcome degree of achievement. Finally, actions were taken to fill in the gaps of the curriculum. Actions were also mapped to the students’ medical outcomes (SOsM).ResultsWeighted averages of obtained quantitative values, mapped to SOsM, indicated accurately the achievement levels of all outcomes as well as the necessary improvements to be performed in curriculum. Mapping the improvements to SOsM also helps in the assessment of the following cycle.ConclusionThe suggested assessment tools can be generalized and extended to any other BME department. Robust improvement of medical content in BME curriculum can subsequently be achieved.

Gait signal classification using an in-house built goniometer and naïve Bayes classifier

This work aims at designing and implementing a knee and an ankle goniometer, both based on potentiometry, and applying the naive Bayes classifier on the signals obtained from the goniometers to differentiate between male and female gait signals, and to also differentiate between healthy and restricted knee gait signals. Gait signals and other parameters were collected from 60 subjects using the goniometers and WEKA was used to classify this data. The designed goniometers were 97.8% accurate and the naive Bayes classifier was highly accurate in categorising the signals with an accuracy of at least 86.7%.

Polydimethyl siloxane microfluidic channel protein functionalization techniques

The most commonly used polymer in microfluidic device fabrication is polydimethylsiloxane (PDMS). Many applications require the functionalization of the surface of PDMS with proteins (e.g. antibodies) which prompted the evaluation of a variety of techniques that have been developed to bind proteins to the surface. such as non-specific binding, protein passive adsorption and activation with Glutaraldehyde cross-linking, or with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC) cross linkers. It has been shown that using EDC crosslinker after APTES activation results in more than double the protein bonding strength to the PDMS surface than the bonding strength that results from passive/non-specific adsorption. The contribution of PDMS curing conditions on protein adsorption to its surface was also studied, and showed that increased curing time is the factor that reduces passive adsorption the most. Finally, we have shown that reaction time between the protein and surface has a direct relationship with bond strength and that initial protein concentration in solution was only an effective factor when the protein was at low concentrations.