new trends in the application of paper based analytical devices

Recently, I went to ACS Fall 2013 National meeting in Indianapolis. I presented two talks. My first talk was about a new microfluidic based ELISA (enzyme linked immunosorbent assay) method for measuring very low concentrations of protein markers and it was in a session called "portable instrumentation for chemical analysis". My second talk was about a new kind of enzyme substrate for ELISA applications and was in a session called "capillary and microfluidic platforms for bioanalytical measurements". Both sessions were under analytical chemistry division. 

As I am more interested in paper based devices these days, I attended talks on low cost analytical devices primarily based on paper. Some talks by professors/students and more posters. I believe the research presented there will be seen more and more in future. Let me point out some of them.

1. paper based devices to monitor low quality pharmaceuticals and iodizing agents in salt.

PBAD used for testing pharmaceuticals. source: ACS

Professor Marya Lieberman and her group from the university of Notre Dame have developed paper based testing devices based on simple colorimetric chemistry. These devices are used to detect low quality pharmaceuticals, iodizing agents in salt etc. They have used these devices in really a low resource settings like in Kenya, Haiti, Iraq, India etc. The idea is that these devices can be sent out to every house/village to test whether the medicine (commonly used) contain what they are supposed to contain. In developing countries this is a very big problem. Prof Toni Barstis's group from Saint Mary's college is also working on paper based colorimetric devices that distinguishes between genuine and low quality anti-malaria ACT (artemisinin combination therapy) medications. The same group has also focused on identifying counterfeit drugs for river blindness.

2. paper based devices for detecting microorganism like bacteria, fungi etc.

One group in Canada are developing paper based culture devices for bacteria. Professor Carey's group from Taiwan are working on single step colorimetric sensing of human pathogenic bacteria in blood.  Their paper device has an array of several dyes (proprietary) that give a signal when reacted from different metabolic by-products of bacteria. This produces kind of finger print to identify bacteria. When successful, this technique could come to market for regular bacteria assays. click here to see their recent publication. Similar to the work by Dr. Carey's group, a group from the University of Illinois at Urbana-Champaign have worked on differentiation and identification of pathogenic fungi based on colorimetric sensor array that reacts with the volatile organic compounds produced from fungi.

Stochastic protein expression in individual cells at the single molecule level

(This was a project report for a microfluidic class written by Ravi Peesera.)

Introduction

Microfluidics is a prominent modern technology which deals with the manipulation of fluids in very small quantities, and has a very large number of potential applications in many fields of science.^2,3 In the field of analysis, microfluidics offers distinct advantages in conferring the ability to handle minute quantities of samples, when coupled with powerful analytical techniques opens up a vast number of applications. Single molecule detection methods are the emerging technologies which carry out measurements at single molecule level, which remove the discrepancies in bulk, ensemble average sample measurements. Microfluidics, with small sample handling capability, makes single molecule measurements possible and further makes the experiments highly designable, in allowing varied designs of miniaturized microfluidic chip systems.

The publication “Stochastic protein expression in individual cells at the single molecule level”¹ is a relevant example of measurements carried out with the combination of microfluidics and single molecule technology, in understanding the events of protein production. The transcription of DNA to messenger RNA and the translation to protein synthesis that follows occurs in a stochastic way in living cells. This randomness makes the development of assays to identify processes during the protein production difficult on a large ensemble of cells, due to the lack of synchronization of these events between cells, thus necessitating the use of single cell level measurements to understand these processes correctly.

β-galactosidase (β-gal) is a hydrolase enzyme that catalyzes the hydrolysis of biologically bound galactosides into galactose monosaccharides in living systems.⁴ Further, β-gal has been shown in various studies to be the standard reporter for gene expression in prokaryotic and eukaryotic cells. In 1961, Rotman and coworkers demonstrated that β-gal is active only as a tetramer and that a single molecule of β-gal can produce a large number of fluorescent product molecules when treated with the synthetic FDG molecule shown in the figure below.⁵ As shown in the figure below, β-gal reacts with the synthetic FDG, which contains two galactose units, cleaves both of them and liberates two molecules of galactose and one molecule of the fluorescent fluoroscein molecule. By virtue of the enzymatic activity β-gal reacts with a large number of FDG molecules, breaks them down by hydrolysis, thus liberating a large number of the fluoroscein molecules. This leads to an accumulation of these fluorescent molecules in the cell which would serve as a marker for the presence and thus the expression of β-gal, which in turn pinpoints to events in protein synthesis. Thus the accurate measurement of this amplified fluorescence in the cell at the single molecule level is crucial to development of an assay to understand this process in real time.

This exiting design of the experiment however has a limitation biologically. After the hydrolysis of FDG’s by the β-gal expressed during protein production, the resulting foreign organic fluoroscein molecules are acted upon by the cellular mechanism and are pumped out of the cell cytoplasm by the efflux pumps on the cell membrane, actively and efficiently. The pumped out fluoroscien molecules diffuse away rapidly into the extracellular region, and so the amplification of the fluorescent signal gained in the cell is lost. So in order to suppress the loss of fluorescence signal, arresting the diffusion process and the localizing it to an area close to the cell is crucial for the measurement of this signal without loss of the enzymatic β-gal amplification.

Theory and Principles

Long Cai and coworkers at Harvard chose to address this problems by using microfluidics to develop an assay to measure the fluorescence without loss of the amplified signal.¹ The cells were trapped in closed microfluidic chambers which ensures that the fluorescent product expelled from the cell is accumulated in small volumes around the cell in these chambers thus stopping the diffusion loss of enzyme amplified fluorescence. The short mixing time of the FDG reagent and the rapid efflux rate at the cellular membrane ensure that the fluorescence signal outside the cells accurately reflects the enzymatic activity happening in the cell. The requisite microfluidic device is made using a soft polymer, polydimethylsiloxane (PDMS), and consist of a flow layer that contains the cells and a top control layer as shown in figure 1. The actuation of the two adjacent valves in the control layer forms an enclosure of the dimensions 100*100*10 µm³ in which the cells are trapped and cultured. The experiment was carried out by mounting the microfluidic chip onto an inverted fluorescence microscope and was translated by a motorized stage, thus allowing multiplexing of the data acquisition by repeated scans on the chamber, carrying out a single scan of 100 chambers in 2 min typically. As shown in figure 1 fluorescence is exited by a tightly focused laser beam that does not directly illuminate the cell, which ensures no cellular auto fluorescence and photo-damage to the cell do not happen.

Figure 1: Schematic diagram of the microfluidic chamber 

used for the enzymatic assay.¹

Initially experiments were carried out to test the system for single molecule measurements. This was done by injecting β-gal enzyme solutions which are very dilute into the chambers and then the fluoregenic fouorescien digalactoside( FDG) substrate was introduced into the chambers. It was observed that the fluorescent signals increase with time, and the slopes of curves shown in the figure 2 give the rates of the hydrolysis.  It was found from these plots that the distribution of hydrolysis rates measured in different chambers showed quantized and evenly spaced peaks which in turn was attributed to the presence of integer numbers of β-gal molecules. Also the spacing between the peaks is 60pMmin^-1., which tells the calibration for the increase in rate of fluoroscein concentration corresponding to one enzyme molecule in the chamber. 

Nanofluidic Platform for Virus Study

Two weeks ago, Prof Jacobson from Indiana University gave an invited talk at our department. His research focuses on developing microfabricated instrumentation and using this instrumentation to study various chemical and biochemical problems. His group is currently pursuing projects which fall into the areas of (1) microfluidic separations, (2) bacteria based studies, (3) fundamentals of nanofluidics, and (4) nanofluidic sensing. 

In his talk he focused primarily on sensing viruses using nanofluidic devices. Fabrication of nanofluidic device itself is challenging. They fabricate nanochannels which connect two microchannels. Shape (angle) of the nanochannel at microchannel-nanochannel junction shows different physical phenomenon. Nanofluidic systems can be significantly influenced by phenomena such as double layer overlap, surface charge, diffusion, and entropic forces, which are either insignificant or absent in larger microchannels. 


They have studied the characterization of Hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. When the virus capsid is allowed to travel through the nanopore, current on the nanopre is monitored. During this event, the conductivity on the nanopore changes because the virus capsid replaces the conductivity solution. This allows to monitor the virus capsid. 


Similar strategies have been used to characterize DNA molecules. DNA molecules are passed through nanochannels and change in current is monitored. When such molecules passe through the pores, there is decrease in current episode.

Advantages of kinetic-ELISA over end point ELISA

Enzyme linked immunosorbant assay (ELISA) is a popular biomedical technique, developed around 40 years back, mainly used in clinical diagnosis to detect disease biomarkers. It takes the advantage of specific interaction between antigen and antibody. The enzyme used in this technique convert substrate molecules into product molecules that are detected in a variety of ways. Because the reaction is enzyme catalyzed, product molecules are generated continuously, it amlifies the assay signal so that low concentrations of analytes can be measured . Basics of ELISA can be found in wiki.

There are two different ways to monitor the assay signal in ELISA. They are end point ELISA and kinetic ELISA. In end point of ELISA, the enzyme reaction is stopped after certain time (usually 30 min) by adding some quenching agents (generally acids or bases or some other enzyme inhibitors) and the signal generated is recorded. This gives just one data point for one sample. Most of the commercial microtiter plate formate ELISA kits follow end point ELISA. But this system has several disadvantages as listed below.

1. Even after the addition of quenching solution the reaction can continue, if the contents are not mixed well.
2. Optical properties of product molecule may change (less colored) by adding acid or base for quenching.
3. Main disadvantage for quantitative analysis is that it has to rely on single data point and this can lead to wrong result.
4. If the background is high, it would give high signal for sample. Also, variations in background signal among the assay chambers lead to inaccurate results.

Most of the disadvantages listed above can be overcome by using kinetic ELISA (k-ELISA). In my PhD research, I am working with kinetic ELISA on microfluidcs chips. In this case, the enzyme reaction is monitored continuously (in our lab weI take readings every 5 min). A graph is plotted as assay time on x-axis and signal on y-axis. The slope of the linear line correspond to the concentration of the analyte. Such linear lines are obtained for each different dilutions or concentrations of sample.  This type of ELISA depends on several data points for each assay which is more quantitative. And also even the background is different, it doesn't really matter. What matters is the slope of the line. It also eliminates quenching step. 

First Microfluidic Chip Ready to Use

As I have started working on microfluidic devices for last couple of weeks, my first chip is ready for use. This is a glass microchip intended to enhance the sensitivity of ELISA. Design of the this chip is not complex. Fabrication of such chips requires more art than science, I believe. My chip is not perfect but good enough to carry out some of the tests that I am planning in following days. I am happy for this. 

The most difficult and uncertain part of chip fabrication was bonding two glass plates-the substrate in which channels were developed and a cover plate to cover the substrate plate. I will write in details about each steps involved in fabrication with my experience in upcoming posts. Keep visiting this blog. In brief photo-lithography and wet etching process were employed and room temperature bonding was used. Below is the picture of my chip.