Analyte preconcentration results in detecting lower concentrations of cancer marker

It is important to detect lower concentrations of disease (like various cancers) markers in human bodily fluids reliably. The ability to detect lower analyte concentration would allow us to identify the onset of diseases in their early stages and start treatment process.

In an effort to make a device that could measure lower concentrations of protein, researchers of Dutta  group at the University of Wyoming have developed a microfluidic device in which the analyte protein molecules are preconcentrated infront of membrane inside the channel. Enzyme Linked Immunosorbent Assay (ELISA) was performed on the same channel and the signal generated from the enzyme reaction was increased by a factor of 200.

In their current work reported, they have shown a reduction in the smallest detectable concentration of the tumor marker CA 199-9 and Blue tongue Viral antibody by over 2 orders of magnitude compared to immunoassays without preconcentration. This led to the improvement in the lowest analyte concentration detectable (LOD) by a factor of 20. Their device only required ~5 uL analyte and other solutions to complete the assay in contrast to conventional microtiter plate based assayed (~100uL).

There are several methods to amplify the ELISA signal and some of them involve preconcentration of enzyme reaction product (e.g resorufin) molecules. Dutta groups new method is unique in terms of preconcentrating target analyte-proteins. They used photopolymerized semipermeable membrane fabricated inside bonded glass microchip for preconcentrating analytes.

Details of this work has been published recently in Analytical Chemistry journal.

Floating Electrodes for fast analyte enrichment

There are several methods for pre-concentration of analyte in microfluidics devices. One of them is using floating electrode. Floating electrode sits inside channel. Normally the electrode is made up of inert metal like gold or platinum. 

Lets have a simple system with a straight channel, two reservoirs at the two end of the channels and the floating electrode sits on the half way inside the channel. When appropriate voltage is applied using platinum electrodes dipped in the buffer solution at two reservoirs, floating electrode receives induced voltage. Negative potential is developed towards anodic region and positive potential is developed towards the cathodic region of the channel. This way floating electrode has two poles. That's why some times it is called as bipolar electrode.

Lately, I am reading papers regarding such electrodes and possible application in ELISA.

The first paper talks about the one-step concentration of different analytes (anionic and cationic) in capillary electrophoresis. They inserted a short platinum wire into the capillary and applied high voltage across two ends of the capillary. It creates a pH gradient along the capillary by the electrolysis of water present. Analytes are concentrated near the platinum wire because of the change in analyte charge which is caused by pH change. They have reported upto several hundredfold concentration factors for anionic analytes (e.g 5(6)-carboxyfluorescein, fluorescein and 2,7-diacetate dichlorofluorescein) and ~30 fold concentration factor for cationic analytes (e.g rhodamine 6G and rhodamine B). They performed separation and concentration at the same time.

Crooks group from The University of Texas at Austin are exploring the use of bipolar electrode for fast concentration enrichment of analytes. They have studied advantages and fundamentals of floating electrode in PDMS microchannel sealed to a glass slide with the gold bipolar electrode at the center.

Crooks group have used BODIPY disulfonate dye in TRIS-HCl buffer. The dye is doubly negatively charged and buffer is positively charged. They have reported that concentration enrichment occurres because of electric field gradient NOT because of pH gradient as described by first paper. In their system, when voltage is applied (~5kV/m) dye is concentrated near to bipolar electrode towards anodic compartment of the channel. Important thing in this system is: above the BPE (bi-polar electrode) current is mainly carried by BPE and ionic current is diminished. This collects dye molecules.

This relatively new way of analyte enrichment could be used in biological applications. I see great future of this method.