David Marshall Porterfield

Frequency-domain fluorescence lifetime optrode system design and instrumentation without a concurrent reference light-emitting diode

We report the design, development, and implementation of an improved instrumentation approach for frequency-domain fluorescence lifetime (FDFL) optrodic sensing without a concurrent reference LED. FDFL traditionally uses a reference LED, at approximately the same wavelength as the sensor fluorophore emission, to measure phase shifts associated with changes in the fluorescence lifetime of fluorophore. For this work we used an oxygen optrode to design, develop, and test the reference-LED-free FDFL approach. Electronics and optics were optimized, and key system parameters, such as inherent system phase shifts, were determined to insure best performance. In our tests with the oxygen optrode, we observed that several key performance characteristics were improved by the implementation of the reference-LED-free instrumentation platform. This system can potentially be adapted to other analyte-selective fluorophores, which will enable scientists and researchers to expand the application of optrodic sensors as basic research tools in biology, medicine, and agriculture.

High-density data acquisition system and signal preprocessor for interfacing with microelectromechanical system-based biosensor arrays

Microelectromechanical system (MEMS) development has become an active area for research in over the last decade. This area has advanced rapidly in recent years due to the potential ability of MEMS devices to perform complex functions in a smaller area. There is also the prospect to develop devices that can (1) be easily manufactured, (2) offer low power consumption, and (3) reduce waste. Especially in the BioMEMS area these advantages are important in terms of applied devices for biosensing, clinical diagnostics, physiological sensing, flow cytometry, and other lab-on-a-chip applications. However, one major obstacle that has been overlooked is the interface of these microdevices with the macroworld. This is critical to enable applications and development of the technology, as currently testing and analysis of data from these devices is mostly limited to generic microprobe stations. New advancements in BioMEMS have to occur in concert with the development of data acquisition systems and signal preprocessors to fully appreciate and test these developing technologies. In this work, we present the development of a cost effective, high throughput data acquisition system (Bio-HD DAQ) and a signal preprocessor for a MEMS-based cell electrophysiology lab-on-a-Chip (CEL-C) device. The signal preprocessor consists of a printed circuit board mounted with the CEL-C device and a 64-channel filter/amplifier circuit array. The data acquisition system includes a high-density crosspoint switching matrix that connects the signal preprocessor to a 16-channel, 18 bit, and 625 kSs DAQ card. Multimodule custom software designed on LABVIEW 7.0 is used to control the DAQ system. While this version of the Bio-HD DAQ system and accompanying software are designed keeping in view the specific requirements of the CEL-C device, it is highly adaptable and, with minor modifications, can become a generic data acquisition system for MEMS development, testing, and application.

Biochips and other microtechnologies for physiomics

This paper presents a review of microtechnologies relevant to applications in cellular physiology, including biochips, electrochemical sensors and optrodic sensing techniques. Microelectrodes have been the main tools for measuring cellular electrophysiology, oxygen, nitric oxide, neurotransmitters, pH and various ions. Optical fiber sensing methods, such as indicator-based optrodes, with fluorescence lifetime measurement, are now emerging as viable alternatives to electroanalytical chemistry. These new optrode techniques are possible because of recent advances in the optoelectronics industry and are comparably easier to miniaturize, have faster response times, do not consume the analyte and have lower operational costs. This review serves as a summary and predicts future trends for both electrochemical and optical luminescence lifetime sensing as components in lab-on-a-chip devices for physiological sensing.

ENHANCING NITROGEN USE EFFICIENCY OF POTATO AND CEREAL CROPS BY OPTIMIZING TEMPERATURE, MOISTURE, BALANCED NUTRIENTS AND OXYGEN BIOAVAILABILITY

Enhancement of nutrient use efficiency is imperative for increasing economic returns and reducing environmental pollution caused by fertilizer use in crop production systems. In this paper, we have demonstrated at a given soil temperature and nitrogen (N) rate, N loss via ammonia emission at 80% field capacity (FC) soil water regime in potato production was decreased by 58 to 81% compared to that at 20% FC, in two soils. In another study, N uptake by flooded corn (genotype: FR27 × FRMO17) seedlings with oxygen fertilization was 8-fold greater than that without oxygen fertilization. Nitrogen utilization efficiency of wheat (cv. ‘Yanzhong 144’) seedlings grown in a complete nutrient solution was 10-fold greater than that of the seedlings under low-phosphorus stress. It is concluded that appropriate management of soil water, oxygen fertilization, and of well-balanced nutrients supply significantly enhance N uptake and utilization efficiencies of corn and wheat, and minimize N loss.

Genotypic differences in potassium nutrition in lowland rice hybrids.

China imports most of its potassium (K) requirements for crop production. The objective of this study was to evaluate indica rice hybrids for K‐use efficiency. Twenty‐eight indica rice hybrids were evaluated in nutrient solution. The K influx rate was greatest in genotype Weiyou 64 (684.9 nmol K+ plant−1 h−1) and least in genotype Xie A/909 (457.2 nmol K+ plant−1 h−1). The K‐use efficiency was greatest in genotype ShanA/909 [81.8 mg dry matter (DM) produced per mg K taken up] and least in genotype Shanyou 64 (55.9 mg mg−1). The maximum biomass was produced by genotype Shan A/4663‐5 (100.8 mg DM per plant), and the least biomass was produced by genotype Xie A/4663‐4 (59.1 mg DM per plant). These results suggest that K shortage for rice production can be alleviated by using K‐efficient rice genotypes.

Amperometric Biosensor Approaches for Quantification of Indole 3-Acetic Acid in Plant Stress Responses

Amperometric biosensors are known to be sensitive, reliable, and inexpensive instruments for biomolecular detection. Recently, self-referencing amperometric biosensors have been utilized to quantify the endogenous apoplastic flux of plant hormone indole 3-acetic acid (IAA) in vivo. There is still a significant need for sampling and testing methods to measure IAA concentrations in whole tissue samples. In the present study we used nanomaterial-modified platinum microelectrodes for the detection of IAA extracted from whole plant tissues. The key to the use of the nanomaterials was to enhance the surface area and thus the limit of detection for IAA. The nanoscale electrochemical interface was modified by the application of a layer of platinum black, followed by silanization overnight and a coating of multiwalled carbon nanotubes. Electroanalytical characterization of sensor performance was evaluated using both IAA standards and IAA extracted from corn plant samples, using a three-electrode scheme (reference, sensing, and auxillary). We compared tissue concentrations in water- and salt-stressed corn seedlings and compared these results to values measured using established enzyme-linked immunosorbent assay (ELISA) protocols. We found that the values obtained from both methods were comparable. The data obtained from the IAA sensor suggested that the electroanalytical biosensor approach was slightly more reliable and sensitive. Our results demonstrate a novel nanomaterial biosensor approach for IAA quantification in plant tissue extracts. The results of the stress study clearly indicated that root and shoot elongation and growth had significant positive correlation with IAA content in the raw plant tissue extracts. Water and salt stress both reduced root and shoot growth, which may be due to overaccumulation of IAA, resulting in inhibition of elongation. In comparison to established methods, such as high-performance liquid chromatography (HPLC) or ELISA, our approach is simple and inexpensive. Unlike HPLC, our approach does not require any elaborate sample purification, nor does it require antibody development as needed for ELISA. In summary, this electroanalytical biosensor method can be effectively utilized for simple, cheap, and reliable detection of IAA extracted from plant samples.

Design, Fabrication and Characterization of an In Silico Cell Physiology lab for Bio Sensing Applications

In this paper, we report the design, fabrication and characterization of an In Silico cell physiology biochip for measuring Ca2+ ion concentrations and currents around single cells. This device has been designed around specific science objectives of measuring real time multidimensional calcium flux patterns around sixteen Ceratopteris richardii fern spores in microgravity flight experiments and ground studies. The sixteen microfluidic cell holding pores are 150 by 150 µm each and have 4 Ag/AgCl electrodes leading into them. An SU-8 structural layer is used for insulation and packaging purposes. The In Silico cell physiology lab is wire bonded on to a custom PCB for easy interface with a state of the art data acquisition system. The electrodes are coated with a Ca2+ ion selective membrane based on ETH-5234 ionophore and operated against an Ag/AgCl reference electrode. Initial characterization results have shown Nernst slopes of 30mv/decade that were stable over a number of measurement cycles. While this work is focused on technology to enable basic research on the Ceratopteris richardii spores, we anticipate that this type of cell physiology lab-on-a-chip will be broadly applied in biomedical and pharmacological research by making minor modifications to the electrode material and the measurement technique. Future applications include detection of glucose, hormones such as plant auxin, as well as multiple analyte detection on the same chip.

Lab-on-a-Chip Approaches for Space-Biology Research

Lab-on-a-chip (LOC) systems with electrochemical sensing capability can provide real-time physiological measurements in spaceflight environments. They are easily miniaturized and integrated with existing space hardware systems. To reduce crew time during spaceflight research, the systems can be made autonomous and simple to use. Research and development of electrochemical-sensing LOC systems are still in progress for fundamental space-biology research in microgravity. Ion-selective electrodes as electrochemical sensors are miniaturized in an all-solid-state format for easier packaging and handling. The design, fabrication, and application of these sensors are discussed, with examples from those developed at the Physiological Sensing Facility (PSF) at Purdue University. The objective of this paper is not to provide an exhaustive review of current LOC systems, but to describe research developments made for the purpose of conducting physiological measurements in microgravity with examples of patents that support space missions.

Ion-Selective Electrode Biochip for Applications in a Liquid Environment

Physiological sensing conducted in a liquid environment requires electrodes with long lifetime. The development of a robust ion-selective electrode–based biochip in a lab-on-a-chip platform is described. To compare electrode lifetime, which is driven by the transducer layer, electrochemical measurements were performed in a custom-made flow-cell chamber. The results of potentiometric measurement of cationic analytes demonstrate the electrodes to have a near-Nernstian slope profile even after they are stored for almost a month in liquid medium. The electrodes also achieved H2O2 amperometric sensitivity (1.25 and 3.32 µAmM−1cm−2 for PEDOT:PSS and PEDOT:CaSO4 respectively) and lower detection limit (2.21 µM, 8.4 µM, 3.44 µM, for H+, NH4 +, Ca2+ respectively) comparable to that of wire-type electrodes. Furthermore, the lifetime is dependent on the electrodeposition method of the conductive polymer, and the transducer layer must be modified to fit the analyte types. These results indicate that extended lifetime of microfabricated ion-selective electrodes in a multiplex format can be realized by optimizing the microfabricated electrode surface functionalization.

In silico cell electrophysiology for measuring transcellular calcium currents

Trans-cellular calcium currents play a central role in the establishment of polarity in differentiating cells. Typically these currents are measured and studied experimentally using ion selective glass microelectrodes. We have recently developed an in silico cell electrophysiology lab-on-a-chip device with the specific science objectives of measuring these transcellular calcium currents in an advanced throughput format. The device consists of 16 pyramidal pores on a silicon substrate with four Ag/AgCl electrodes leading into each pore on the four poles. An SU-8 layer is used as the structural and insulating layer and a calcium ion selective membrane is used to impart ion selectivity to the Ag/AgCl electrodes. In this paper we demonstrate the utility of the cell electrophysiology biochip in measuring these transcellular calcium currents from single cells using the model biological system Ceratopteris richardii. We monitored these fern spores during germination and pharmacologically inhibited biophysical calcium transport. These results demonstrate the utility and versatility of the in silico cell electrophysiology biochip. While this version of the biochip was engineered to fulfill the specific science objectives of measuring trans-cellular calcium currents from Ceratopteris fern spores, the chip can easily be modified for a variety of biomedical and pharmacological applications. Future