Friday, March 13, 2015

Iodometric titration on paper device #PADs

Titration technique is widely used in various analytical service providing and teaching laboratories. In this technique, a solution of known concentration is used to determine the concentration of an unknown sample. The currently used titration methods consume large volume of reagents and samples (hundreds of milliliter) and glassware like burette and pipettes.
Design of iodometric paper test card (source)

Titrations now can be carried out in a piece of paper modified with appropriate reagents. Professor Lieberman's group from University of Notre Dame recently has described an iodometric titration method in a paper card-published in Analytical Chemistry journal. Titration in the paper test card starts by applying a test solution to the test card in which multiple dried reagents have been stored separately. The reagents reconstitute and combine through a surface-tension enabled mixing (STEM) after the application of unknown solution. The end point of the titration is indicated by the appearance of blue complex of iodide and starch. The signal can also quantitated by using image-processing software.

Iodometric titration involves a redox reaction used to determine the amount of variety of analytes of interests. The authors used this method to quantitate the iodine present in common salt and also demonstrated the versatility of the titration device by quantifying beta-lactam antibiotics via an iodometric back-titration. In later example, the antibiotic was degraded in base to obtain a redox active thiol. The reaction mixture was then acidified and a known amount of excess triiodide is added to oxidize the thiol. Ureacted triiodide is back-titrated with thiosulfate.

Unique feature of the iodometric test card is its ability to store multiple reagents separately for long time and allowing them to mix and react when desired.

The paper titration technique would be very useful in developing countries and in remote locations-especially. It is cheap, easy to perform and requires small amount of reagents. Also, generates less waste. Authors described the stability of this test card (stored reagents) for ~20 days at temperature 40deg C. It is not clear if the results obtained at above conditions will be valid for more than 20 days and higher temperature combined with humidity. Lets consider a village in India. Some of the Indian villages see temperatures more than 40 deg C (easily 45) that combined with high percentage of humidity. Will the test card work in this location? Proper packaging can protect effect of humidity, however. If the test card is not manufactured at local level, then 20 days time frame is not enough. It will take months for the cards to be used in field.

This method has great scope to be introduced in to the classroom-teaching labs.
Will this new method replace traditional ways of doing iodometric titrations?

Sunday, January 25, 2015

Acid-base titration on paper microfluidic device: will it replace traditional acid-base titration?

u-PAD for acid-base titration (source)
Research in paper-based microfluidic analytical devices (u-PADs) has evolved very fast in past couple of years. Recently, scientists from Okayama University, Japan have reported a technique to carry out acid-base titrations in PADs and the work has been published in journal Analytical Chemistry. 

The wax printed u-PADs consisted of ten reaction (acid-base reaction) and 10 detection reservoirs. The detection reservoirs were applied with a constant amount of phenolphthalein (indicator) and the reaction reservoirs were applied with various amount of a primary standard potassium hydrogen phthalate (KHPth). Only less than a microliter volume of the solutions were needed. The base (e.g., NaOH) was dropped onto the center reservoir of the device. As the base wicked towards the reaction reservoir, it then reacted with the acid and if any extra NaOH remained it then moved further and reached to the detection reservoir to produce a pink color. The number of detection reservoirs with no color change was used to determine the concentration of NaOH. The authors have tested this technique using other acids like nitric acid, hydrochloric acid, sulfuric acid, and acetic acid.

Acid-base titration is widely used in teaching chemistry  and in variety of analytical services. The currently used titration methods involve large volume of reagents and samples; glassware (burette and pipettes).
The new u-PAD being used in field testing (source)
The new method takes only about a minute to complete the titration and is more advantageous than classical titration methods in terms of speed, portability, and disposability.

Eventhough the micro-titration device was shown to be stable for couple of days, it requires further improvements to make the device stable for longer time. The reagents stored in the PADs degrade with time. Therefore the reagents have to be applied at the time of use only. Also, temperature plays bad role for the stability of the PADs. Therefore not suitable for places with higher ambient temperature (e.g., summer time in India).

As PADs are cheap, this new titration technique may replace the traditional titrations, specially in developing countries. Teaching settings can immediately adopt the technique.

Wednesday, July 16, 2014

How does Google Contact Lens Work?

A prototype Google Contact Lens as Glucose sensor [source]
The Google contact lens is coming to reality. Google and Novartis announced on Monday that they will work together to bring this new technology to the public. Google[x] of Google has developed this technology, unveiled at first in January, that is supposed to measure the levels of glucose in tear. If everything goes as planned, we will see prototype product for research and development reviews by 2015.

Cartoon showing lens components
The soft contact lens consists of a wireless chip and a miniaturized glucose sensor housed in between two layers of lens material. The lens also features a tiny antenna, capacitor, and controller. Data is relayed via wireless antenna that is thinner than human hair.The information gathered from the lens can move from the eye to a device such as a handheld monitor, where that data can be read and analysed.  

This is how the lens looks like.

There is a tiny pinhole in the lens. Tear fluid is seeped into the sensor which measures the glucose level. The electronics lie outside of both the pupil and the iris so there is no damage to the eye. The miniaturized sensor is powered by a wireless device which will communicate data via the wireless technology radio frequency identification (RFID). This wireless non-contact technology uses radio-frequency electromagnetic fields to transfer data. 

This method will be an easier, pain free, and non-invasive alternative to currently used methods that require prick fingers for droplets of blood. The new technology can give you reading every second. The developers of this technology are also planning to add small LED lights that could warn you when the glucose levels cross certain threshold level that are critically harmful.

Apps developed for this technology would make the measurements available to the wearer and their doctor.

Google will ensure that any data transferred from the lens cannot be manipulated. 

More readings:

Thursday, July 10, 2014

How to make glass microfluidics device? chemical wet etching and room temperature bonding

[First part of this post is here where I wrote step by step procedure for mask design and photolithography.]

After completing the photolithographic procedure, we use two different solutions for selectively removing chromium and glass from the channel network on the bottom substrate. 

Chemical wet etching solutions. On left is BOE with temperature control
and on right is Chrom etchant. Middle one is DI water.
At first, chromium layer from channel network is removed by immersing the substrate plate into a Teflon coated jar containing chromium etchant solution for about 15 min. One should note that leaving substrate plate in this solution for long time (hours and hours-this happens when you forget to remove it) could eat away even the photoresist layer from the entire plate. We buy the chrome etchant from Transene Company, INC, MA, USA and according to manufacturer specification the solution contains ceric sulfate (5-10%), nitric acid (5-10%), sulfuric Acid (1-5%), water (~ 75%). The entire substrate is then washed with DI water and dried by blowing N2 gas. At this point one can clearly see through the substrate in channel patterning region.
Cartoon depicting the removal of photoresist and chromium layer from channel pattern

After this step, we cut the substrate into individual chips (1" x 2") using a glass-cutter (altogether 8 chips from one 8” x 8” substrate). Then, put extra layer of photoresist, dry in oven (80 o C), take out from the oven, and let it cool.

The substrate plate-individual chip is then immersed into a 1:10 buffered oxide etchant (BOE) solution bought from Transene Company, INC, MA, USA. According to the company specifications, this solution contains a mixture of hydrofluoric (HF) (1-25%), ammonium fluoride (NH4F) (2-40%), distilled water. Hydrofluoric acid is a hazardous chemical, therefore must be handled with extra precaution. The BOE solution is continuously stirred at the rate of 120 rpm using a magnetic stirrer and the temperature is set at 55 oC. The substrate plate is removed from the BOE solution at a certain interval of time ~10-15 min (depending on the etching rate of BOE and channel depth desire), washed with DI water, and is dried with N2. With our settings, etching rate is ~0.5 micron per minute. 

However, this rate can be manipulated based on your need. 
Cartoon showing the substrate plate after removing glass material.
Remember the glass etching is in all direction (not like in this cartoon)

The depth of the channel is measured using a XP series stylus profiler (Ambios Technology, CA, USA). This step is repeated until one gets desired channel depth.  

Once desired channel depth is obtained, ~1 mm diameter access holes were punched at the channel terminals using a microabrasive power blasting system (Vaniman) by blowing sand particles from back side of the substrate. The miroabrasive system utilizes mechanical erosion on the substrate by bombarding with high-kinetic energy sand particles and results in conically shaped holes. This step is followed by removing the photoresist and chromium layer on remaining parts of the bottom substrate by using acetone and chromium etchant, respectively. 
Bonding two glass plates: The etched channels in the bottom substrate were sealed using cover plate. The two plates (bottom substrate and cover plate) were cleaned and brought together in a beaker containing DI water for bonding. They were removed from the beaker together with a tweezer, water was removed by gently pressing the plates with hand in paper towel, and then put under couple of heavy books for ~2 hrs. Then the bonded chip was kept in an oven at 80 oC for ~1hrs to strengthen the bonding between two glass plates. Bonding is a crucial step to create closed fluidic microchip networks. 

The bonding strength between two surfaces is proportional to the density of individual chemical bonds established between the two plates. Our bonding method is simple and does not require clean room facilities, programmed high-temperature furnaces, pressurized water sources, and adhesives. The bonding process can be completed in ~3 hrs. One of the most important factors affecting successful bonding of planner glass chips is the cleanliness of the bonding surfaces of glass substrates. Unsuccessful bonding events are often associated with solid particles or organic material remaining on the glass surfaces, for example, dust, residual photoresist, chromium or glass particles etc., prior to bonding. Some other critical factors for glass bonding are flatness of the glass plates and chip area.

Monday, May 5, 2014

Can Google glass test HIV and prostate cancer? #newtechnology

Google Glass taking image of RDT
A Google Glass-based platform has been shown to detect human immunodeficiency virus (HIV) and prostate-specific antigen (PSA). Since its limited availability to public, Google Glass has been used for variety of applications.
The GoogleGlass is famous for a voice-controllable hands-free computing system that can perform imaging and video-recording tasks. In addition, various wireless technologies including Bluetooth and Wi-Fi are available in it. It can access internet and determine location information via global positioning systems (GPS) and/or triangulation through cellular service provider towers.
Google Glass @ACSNANO
The details of recently demonstrated Google Glass-based rapid diagnostic test (RDT) reader platform has been published in American Chemical Society journal ACSNANO by a group of scientists from University of California, LA, USA. This platform was used in lateral flow immunochromatographic assays that are important in biomedical diagnostic tests.

Saturday, February 15, 2014

How to make glass microfluidic device? mask design and photolithography

During my PhD at the University of Wyoming, I may have made ~400-500 microfluidic devices all from glass substrate. My PhD laboratory uses borosilicate based substrate/cover plate to make the chips for variety of applications such as immunoassay development, separation experiments, fuel cell units. Glass is one of the oldest materials in microfluidic field and has certain advantages (see below) over other materials that are being used in this field of research.
  1. Inertness to many chemicals
  2. Optical transparency
  3. Low fluorescence
  4. High resistance to mechanical stress
  5. Well established surface modification procedures
Below are the steps taken for the fabrication of glass microfluidic device: starting from designing the channel network using CADopia software to bonding the glass plates. I will take you through each of these steps.

1. Mask design: We create a channel design using CAPopia drawing software. I had never used any drawing softwares before and found this one easy to use. Once the design is complete, we send it out to a printing company (Fineline Imaging, Colorado) to get the mask. 

Mask with channel design

2. Photolithographic procedure: Once the photo-mask arrives from the printing company, we perform basic photo-lithography procedure to transfer the channel pattern design onto the glass substrate.  We buy borosilicate glass substrate (Telic, CA, USA) which is 4” x 4” in dimension and 1.65 mm thick. One side of this glass substrate is coated with a layer of chromium and photoresist on top of each other. Both chromium and photoresist layers are about 100-200 nm in thickness. The photoresist coated on the substrate is polymer based positive photoresist which is sensitive to UV radiation. Therefore the box containing the substrate plates must be opened only in dark room.

In photolithography room turn on the UV light source before at least 15 min you plan to shine it to the substrate plate. We use a custom made box where one can place photo-mask on top of substrate plate. This assembly is then gently pressed by putting a glass plate on top of it. The whole thing is inside a box in which a shutter can be opened when ready to expose to UV radiation.

Typically we expose the substrate to the UV radiation for ~ 30-45sec. During this step the polymer of photoresist breaks down and is removed by soaking the substrate in a photo-developing solution (MF-319, Rohm and Haas) for ~ 5 min. The substrate is then washed with deionized (DI) water (DirectQ Water Purification System, Millipore) and dried by blowing N2 gas. After this washing step, one can clearly see the channel pattern on the glass substrate. 

click HERE for second part of this post

Thursday, January 30, 2014

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.