14 research outputs found
DStat: A Versatile, Open-Source Potentiostat for Electroanalysis and Integration
<div><p>Most electroanalytical techniques require the precise control of the potentials in an electrochemical cell using a potentiostat. Commercial potentiostats function as “black boxes,” giving limited information about their circuitry and behaviour which can make development of new measurement techniques and integration with other instruments challenging. Recently, a number of lab-built potentiostats have emerged with various design goals including low manufacturing cost and field-portability, but notably lacking is an accessible potentiostat designed for general lab use, focusing on measurement quality combined with ease of use and versatility. To fill this gap, we introduce DStat (<a href="http://microfluidics.utoronto.ca/dstat" target="_blank">http://microfluidics.utoronto.ca/dstat</a>), an open-source, general-purpose potentiostat for use alone or integrated with other instruments. DStat offers picoampere current measurement capabilities, a compact USB-powered design, and user-friendly cross-platform software. DStat is easy and inexpensive to build, may be modified freely, and achieves good performance at low current levels not accessible to other lab-built instruments. In head-to-head tests, DStat’s voltammetric measurements are much more sensitive than those of “CheapStat” (a popular open-source potentiostat described previously), and are comparable to those of a compact commercial “black box” potentiostat. Likewise, in head-to-head tests, DStat’s potentiometric precision is similar to that of a commercial pH meter. Most importantly, the versatility of DStat was demonstrated through integration with the open-source DropBot digital microfluidics platform. In sum, we propose that DStat is a valuable contribution to the “open source” movement in analytical science, which is allowing users to adapt their tools to their experiments rather than alter their experiments to be compatible with their tools.</p></div
Cell current conversion to voltage for ADC.
<p>(a) Current measurement by shunt resistor. The measurement resistor <i>R</i><sub><i>M</i></sub> causes a voltage drop proportional to the cell current <i>i</i> by Ohm’s Law. The voltage drop is measured across the resistor but the counter electrode voltage <i>V</i><sub><i>CE</i></sub> (present on both sides of the resistor) complicates measurement. (b) Current measurement using a transimpedance amplifier. The measurement resistor <i>R</i><sub><i>M</i></sub> is placed in a negative feedback loop of an op amp (U3) whose inverting input is connected to the working electrode. U3’s non-inverting input is tied to ground, producing a virtual ground at the inverting input. When current <i>i</i> flows through the working electrode, it induces a voltage drop <i>V</i><sub><i>R</i></sub> across <i>R</i><sub><i>M</i></sub>, which is balanced by U3 output <i>V</i><sub><i>O</i></sub> to maintain the virtual ground at its inverting input.</p
The DStat.
<p>(a) Schematic overview of key DStat components, including the computer (PC), the microcontroller (XMEGA), the analogue-to-digital converter (ADC), the digital-to-analogue converter (DAC), the potentiostatic circuit, and transimpedance amplifier. The DStat is interfaced to a three-electrode electrochemical cell, including a working electrode (WE), a counter electrode (CE), and a reference electrode (RE). Modules integral to DStat are coloured in green. Solid lines represent analogue connections. Dotted arrows represent digital connections. (b) Top-view picture of the DStat circuit board with labels corresponding to schematic components.</p
DStat/Dropbot integration.
<p>Solid arrows represent communication between the control computer and instruments over USB and dotted arrows represent communication between programs within the control computer over ZeroMQ. (a) When Dropbot’s control software μDrop reaches a programmed electrochemical measurement step, it pauses droplet actuation (in the figure, represented by a droplet parked at a circular electrochemical cell similar to the one described by Dryden et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140349#pone.0140349.ref031" target="_blank">31</a>]) and requests an electrochemical measurement from the DStat software. The DStat software processes the request by initiating an experiment on the DStat hardware. (b) As the DStat hardware performs the experiment, the DStat software records the data. When the experiment is complete, the DStat software reports to μDrop that the measurement is complete and μDrop resumes its programmed droplet movement. A movie depicting the full process can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140349#pone.0140349.s006" target="_blank">S1 Video</a>.</p
Electrochemical cells and potentiostatic circuits.
<p>RE: Reference Electrode, CE: Counter Electrode, WE: Working Electrode, R: Summing resistors, U1: Control amplifier, U2: Reference buffer amplifier, <i>R</i><sub><i>c</i></sub>: Compensated cell resistance, <i>R</i><sub><i>u</i></sub>: Uncompensated cell resistance. (a) Simplified three electrode cell model. (b) Basic potentiostatic circuit. (c) DStat potentiostatic circuit.</p
Digital Microfluidic Platform for Human Plasma Protein Depletion
Many important biomarkers for disease
diagnosis are present at
low concentrations in human serum. These biomarkers are masked in
proteomic analysis by highly abundant proteins such as human serum
albumin (HSA) and immunoglobulins (IgGs) which account for up to 80%
of the total protein content of serum. Traditional depletion methods
using macro-scale LC-columns for highly abundant proteins involve
slow separations which impart considerable dilution to the samples.
Furthermore, most techniques lack the ability to process multiple
samples simultaneously. We present a method of protein depletion using
superparamagnetic beads coated in anti-HSA, Protein A, and Protein
G, manipulated by digital microfluidics (DMF). The depletion process
was capable of up to 95% protein depletion efficiency for IgG and
HSA in 10 min for four samples simultaneously, which resulted in an
approximately 4-fold increase in signal-to-noise ratio in MALDI-MS
analysis for a low abundance protein, hemopexin. This rapid and automated
method has the potential to greatly improve the process of biomarker
identification
Digital Microfluidic Platform for Human Plasma Protein Depletion
Many important biomarkers for disease
diagnosis are present at
low concentrations in human serum. These biomarkers are masked in
proteomic analysis by highly abundant proteins such as human serum
albumin (HSA) and immunoglobulins (IgGs) which account for up to 80%
of the total protein content of serum. Traditional depletion methods
using macro-scale LC-columns for highly abundant proteins involve
slow separations which impart considerable dilution to the samples.
Furthermore, most techniques lack the ability to process multiple
samples simultaneously. We present a method of protein depletion using
superparamagnetic beads coated in anti-HSA, Protein A, and Protein
G, manipulated by digital microfluidics (DMF). The depletion process
was capable of up to 95% protein depletion efficiency for IgG and
HSA in 10 min for four samples simultaneously, which resulted in an
approximately 4-fold increase in signal-to-noise ratio in MALDI-MS
analysis for a low abundance protein, hemopexin. This rapid and automated
method has the potential to greatly improve the process of biomarker
identification
Digital Microfluidic Magnetic Separation for Particle-Based Immunoassays
We introduce a new format for particle-based immunoassays
relying
on digital microfluidics (DMF) and magnetic forces to separate and
resuspend antibody-coated paramagnetic particles. In DMF, fluids are
electrostatically controlled as discrete droplets (picoliters to microliters)
on an array of insulated electrodes. By applying appropriate sequences
of potentials to these electrodes, multiple droplets can be manipulated
simultaneously and various droplet operations can be achieved using
the same device design. This flexibility makes DMF well-suited for
applications that require complex, multistep protocols such as immunoassays.
Here, we report the first particle-based immunoassay on DMF without
the aid of oil carrier fluid to enable droplet movement (i.e., droplets
are surrounded by air instead of oil). This new format allowed the
realization of a novel on-chip particle separation and resuspension
method capable of removing greater than 90% of unbound reagents in
one step. Using this technique, we developed methods for noncompetitive
and competitive immunoassays, using thyroid stimulating hormone (TSH)
and 17β-estradiol (E2) as model analytes, respectively. We show
that, compared to conventional methods, the new DMF approach reported
here reduced reagent volumes and analysis time by 100-fold and 10-fold,
respectively, while retaining a level of analytical performance required
for clinical screening. Thus, we propose that the new technique has
great potential for eventual use in a fast, low-waste, and inexpensive
instrument for the quantitative analysis of proteins and small molecules
in low sample volumes
Digital Microfluidics for Immunoprecipitation
Immunoprecipitation
(IP) is a common method for isolating a targeted
protein from a complex sample such as blood, serum, or cell lysate.
In particular, IP is often used as the primary means of target purification
for the analysis by mass spectrometry of novel biologically derived
pharmaceuticals, with particular utility for the identification of
molecules bound to a protein target. Unfortunately, IP is a labor-intensive
technique, is difficult to perform in parallel, and has limited options
for automation. Furthermore, the technique is typically limited to
large sample volumes, making the application of IP cleanup to precious
samples nearly impossible. In recognition of these challenges, we
introduce a method for performing microscale IP using magnetic particles
and digital microfluidics (DMF-IP). The new method allows for 80%
recovery of model proteins from approximately microliter volumes of
serum in a sample-to-answer run time of approximately 25 min. Uniquely,
analytes are eluted from these small samples in a format compatible
with direct analysis by mass spectrometry. To extend the technique
to be useful for large samples, we also developed a macro-to-microscale
interface called preconcentration using liquid intake by paper (P-CLIP).
This technique allows for efficient analysis of samples >100Ă—
larger than are typically processed on microfluidic devices. As described
herein, DMF-IP and P-CLIP-DMF-IP are rapid, automated, and multiplexed
methods that have the potential to reduce the time and effort required
for IP sample preparations with applications in the fields of pharmacy,
biomarker discovery, and protein biology
Analysis on the Go: Quantitation of Drugs of Abuse in Dried Urine with Digital Microfluidics and Miniature Mass Spectrometry
We report the development of a method
coupling microfluidics and a miniature mass spectrometer, applied
to quantitation of drugs of abuse in urine. A custom digital microfluidic
system was designed to deliver droplets of solvent to dried urine
samples and then transport extracted analytes to an array of nanoelectrospray
emitters for analysis. Tandem mass spectrometry (MS/MS) detection
was performed using a fully autonomous 25 kg instrument. Using the
new method, cocaine, benzoylecgonine, and codeine can be quantified
from four samples in less than 15 min from (dried) sample to analysis.
The figures of merit for the new method suggest that it is suitable
for on-site screening; for example, the limit of quantitation (LOQ)
for cocaine is 40 ng/mL, which is compatible with the performance
criteria for laboratory analyses established by the United Nations
Office on Drugs and Crime. More importantly, the LOQ of the new method
is superior to the 300 ng/mL cutoff values used by the only other
portable analysis systems we are aware of (relying on immunoassays).
This work serves as a proof-of-concept for integration of microfluidics
with miniature mass spectrometry. The system is attractive for the
quantitation of drugs of abuse from urine and, more generally, may
be useful for a wide range of applications that would benefit from
portable, quantitative, on-site analysis