Nuclear Magnetic Resonance (NMR) compatible platform for the automated real-time monitoring of biochemical reactions using a flow shuttling configuration. This platform requires a working sample volume of ∼11 mL and it can circulate samples with a flow rate of 28 mL/min, which makes it suitable to be used for real-time monitoring of biochemical reactions. Another advantage of the proposed low-cost platform is the high spectral resolution. As a proof of concept, we acquire 1H NMR spectra of waste orange peel, bioprocessed using Trichoderma reesei fungus, and demonstrate the real-time measurement capability of the platform. The measurement is performed over more than 60 h, with a spectrum acquired every 7 min, such that over 510 data points are collected without user intervention. The designed system offers high resolution, automation, low user intervention, and, therefore, time-efficient measurement per sample.
Manuscript: A Nuclear Magnetic Resonance (NMR) Platform for Real-Time Metabolic Monitoring of Bioprocesses
Introduction
We demonstrate real-time monitoring of a bioreaction by NMR spectroscopy while using a flow shuttling system to deliver the sample to and from a bioreactor placed outside of the NMR magnet. Our platform enables automated NMR data acquisition by including an interface between the shuttling system and the NMR spectrometer to synchronize sample delivery and data acquisition. The flow cell is a concentric glass tube design, allowing for an internal standard solution to be separated in the outer volume from the sample flowing through the central capillary.
Experimental setup
1) The personal reaction station (PRS) is used to carry out the bioreaction.
2) The sample was regularly transported through the capillary-based flow cell (inside the NMR magnet) for NMR measurement and then returned to the bioreactor using a low-cost peristaltic pump and custom microcontroller-based circuitry, which consists of Arduino Board and a driver circuit.
3) The pumping of the material through the NMR apparatus and the timing of the measurements were both programmed using an Arduino microcontroller.
4) The peristaltic pump's DC motor was driven by an L293 Driver, which was part of the driver circuit.
5) After starting the reaction in the PRS, the resultant solution was pumped through the apparatus by the peristaltic pump and into the NMR measurement zone.
6) The controller provided a TTL (transistor-transistor logic) signal that was used to activate the NMR data acquisition.
2) The sample was regularly transported through the capillary-based flow cell (inside the NMR magnet) for NMR measurement and then returned to the bioreactor using a low-cost peristaltic pump and custom microcontroller-based circuitry, which consists of Arduino Board and a driver circuit.
3) The pumping of the material through the NMR apparatus and the timing of the measurements were both programmed using an Arduino microcontroller.
4) The peristaltic pump's DC motor was driven by an L293 Driver, which was part of the driver circuit.
5) After starting the reaction in the PRS, the resultant solution was pumped through the apparatus by the peristaltic pump and into the NMR measurement zone.
6) The controller provided a TTL (transistor-transistor logic) signal that was used to activate the NMR data acquisition.
Working
1) The temperature inside the personal reaction station is controllable and it was set to the optimal temperature for the fungal growth.
2) The medium of the bio-reaction flowed through the system, for 47 s, which, at a flow rate of 28 mL/min., corresponds to ∼22 mL of solution transported (which was sufficiently larger than the required volume of 10.6 mL of the transport system).
3) After a relaxation time of 50 s, the microcontroller sent a TTL signal to the NMR spectrometer in order to begin the data acquisition.
4) After NMR data acquisition, the microcontroller automatically transported the sample back to the bio-reaction, i.e., the transport lines were emptied.
5) Each repetition of flow, relaxation, and acquisition took approximately 7 min.; hence, every 7 min., a new spectrum of the solution was acquired.
6) In total, 516 NMR experiments were obtained within three days of the experiment.
2) The medium of the bio-reaction flowed through the system, for 47 s, which, at a flow rate of 28 mL/min., corresponds to ∼22 mL of solution transported (which was sufficiently larger than the required volume of 10.6 mL of the transport system).
3) After a relaxation time of 50 s, the microcontroller sent a TTL signal to the NMR spectrometer in order to begin the data acquisition.
4) After NMR data acquisition, the microcontroller automatically transported the sample back to the bio-reaction, i.e., the transport lines were emptied.
5) Each repetition of flow, relaxation, and acquisition took approximately 7 min.; hence, every 7 min., a new spectrum of the solution was acquired.
6) In total, 516 NMR experiments were obtained within three days of the experiment.
FAQs
1. What is NMR technique ?
Ans: Nuclear Magnetic Resonance (NMR) spectroscopy is an important analytical technique in both material and medical sciences for real-time investigations of the molecular composition of bioreactions.
2. What is the use of PRS ?
Ans: It provided precise temperature control via a PID controller. In cases where reaction stirring is important, the PRS features magnetic stirring for each reaction tube; for our system, active stirring was not used to avoid destruction of the fungal mycelia
3. What is the purpose of Microcontroller circuitry?
Ans: It provided a TTL signal to activate NMR data capture which further synchronizes sample transport and measurements. It also controls flow rate and direction of sample transport.
4. What can be improved to increase the sensitivity of the system ?
Ans: There are also features to be improved: The transport lines are currently not insulated and, thus, temperature variation is possible. The transfer lines were relatively long, which, if shortened, could significantly reduce the sample transfer times. Our system could not take advantage of the standard NMR lock and auto-shim functions and thus manual intervention was necessary to correct the magnetic field homogeneity.With the lock enabled, solvent suppression could be additionally used in order to suppress the residual water signal and further enhance sensitivity.
Ans: Nuclear Magnetic Resonance (NMR) spectroscopy is an important analytical technique in both material and medical sciences for real-time investigations of the molecular composition of bioreactions.
2. What is the use of PRS ?
Ans: It provided precise temperature control via a PID controller. In cases where reaction stirring is important, the PRS features magnetic stirring for each reaction tube; for our system, active stirring was not used to avoid destruction of the fungal mycelia
3. What is the purpose of Microcontroller circuitry?
Ans: It provided a TTL signal to activate NMR data capture which further synchronizes sample transport and measurements. It also controls flow rate and direction of sample transport.
4. What can be improved to increase the sensitivity of the system ?
Ans: There are also features to be improved: The transport lines are currently not insulated and, thus, temperature variation is possible. The transfer lines were relatively long, which, if shortened, could significantly reduce the sample transfer times. Our system could not take advantage of the standard NMR lock and auto-shim functions and thus manual intervention was necessary to correct the magnetic field homogeneity.With the lock enabled, solvent suppression could be additionally used in order to suppress the residual water signal and further enhance sensitivity.