ELISA with 100 fold improvement in detection limit & 100 fold less sample volume

My first paper on microfluidic work has been published in Analitica Chimica Acta. This was the first project I worked on for my PhD work (actually the very first one didn't work. That was my advisor's crazy idea he wanted me to give a try.  We abandoned that later on). 

In this article, we have demonstrated a novel approach to enhancing the sensitivity of enzyme-linked immunosorbent assays (ELISA) through pre-concentration of the enzyme reaction product (resorufin/4- methylumbelliferone) in free solution. 

Highly sensitive analytical techniques are required to estimate small amounts of disease markers (e.g., antibodies/antigens) in bodily fluids in order to detect the onset of dangerous diseases like cancers at their early stages. Here is the abstract of this paper. 

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. 

ELISA on Microfluidics channels

ELISA (enzyme linked immunosorbant assay) methods are being developed on microfluidics in our lab. My projects involve improving sensitivity of ELISA using microfluidic platform. We fabricate and use glass based microfluidic chips. I have seen some papers which describe ELISA on PDMS chips. The glass chips are cheaper to fabricate, does not require sophisticated instrumentations. We use photolithography, chemical wet etching and room temperature bonding technology to fabricate glass microfluidic chips. I feel I am good at fabricating chips. I don't have any problems till now from the beginning on fabrication. Actually I learned this step quickly.  After being able to fabricate chips, I started doing static ELISA. Here comes the problem. Its being two weeks I am not able to get expected data.

ELISA in microchannel involves different coating steps. First, channels are cleaned with sodium hydroxide to form negatively charged-hydrophilic  glass surface. We use horse redish peroxide (HRPase) as enzyme which converts amplex red (fluorophore) to resorufin (a fluorescent anionic molecule). After completing appropriate ELISA steps, amplex red containing hydrogen peroxide is introduced into the channel and the fluorescent signal of resorufin molecules produced by enzymatic conversion of amplex red is monitored.

Fluorescent signal should correspond to the concentration of antigen in the sample. At this point in my experiments, the signal does not increase with the increase in the concentration of analyte. I repeated the same experiment 4 times and got the same result. I might be repeating the same stupid mistake again and again. There must be a systemic error which I am not able to figure out till now.

If you have any clue, advise plz let me know. I would be very great full.

Thank you for reading my experience with ELISA.