Electrochemical Sensing For Biomarker Release Testing

The aim of this project is to develop electrochemical sensing for point of care testing for the release of biomarkers from cells. The biomarker being used is alkaline phosphatase (ALP). An investigation will be carried out to test the cytoxic effect that aloeson, a component of Aloe Vera has on the Ht-29 colon cancer cell lines using optical detection methods. Electrochemical sensing measures the concentration of the target molecule by oxidising or reducing the target at an electrode which results in and measures the resulting electric current. Electrochemistry is the relationship between chemical reactions and electricity.

There are two main ways the electricity and chemical reactions interact (1) certain chemical reactions can create electricity, (2) Electricity can make certain reactions happen that wouldn’t happen otherwise. Electrochemistry has many applications in industry, primarily the production of batteries. Several different methods of electrochemical sensing are used in clinical laboratories such as (1) potentiometry, (2) voltammetry/amperometry, (3) conductometry, and (4) coulometry. * The aim of this experiment is to be able to detect the biomarker ALP by electrochemical sensing. (1) “Potentiometry is the measurement of an electrical potential difference between two electrodes in an electrochemical cell. ”* Potentiometry is widely used for the measurement of pH, PCO2 and electrolytes. There are different types of electrodes used for this technique; redox, ion-selective and PCO2 electrodes.

Potentiometry with ion-selective electrodes (ISEs) means pH is primarily measured with a glass electrode. This technique has revolved over the past 10 years in areas such as environmental trace analysis and potentiometric biosensing. 1 (2) Voltammetry/amperometry is a different form of electrochemical measuring where the methods are based on electrolytic electrochemical cells, where external voltage is applied to polarizable working electrode and the resulting cathodic and anodic current is proportional to the concentration of the analyte being tested in the sample. *(3) Conductometry measures the concentration of the analyte being testing by measuring ty the effect of the electrical conductivity of the mixture.

It measures how well ions can carry an electric current under the influence of potential difference. A clinical application of this is the electronic counting of blood cells in suspension. *(4) Coulometry measures the electrical charge passing between two electrodes in an electrochemical cell*. The amount of charge is directly proportional to the oxidation or reduction of an electroactive substance at one of the electrodes. This can be measured using Faraday’s Law. Q = the quantity of charge passing through the electrochemical cell, n = the number of electrons transferred during the oxidation or reduction process, N = the amount of moles oxidised or reduced and F = faraday constant (96487 coulombs/mole). Optical detection will be the primary detection method used to investigate the cytoxic effect of aloe Vera extract aloesin on the Ht-29 colon cancer cell line. Optical chemical sensors are referred to as an “optode”. Optodes are used to measure blood, gases and electrolytes. Optodes have many advantages over using electrodes such as not needing a reference electrode, stability, and less electric noise.

Light is passed from the sensing sight through optical fibres or by using light sources such as LED, appropriate filters and detectors such as photomultiplier tube (PMT) to monitor absorbance, fluorescence or phosphorescence. Optodes have been created for the measurement of PO2 levels by the immobilization of particular organic dyes; (1) pyrene, (2) diphenylphenathrene, (4) fluoranthene or (5) metal ligand complexes. *The presence of paramagnetic (unpaired) species causes quenching at a certain wavelength of fluorescence or phosphorescence. The Stern-Volmer equation for quenching is given:. * I0 is the intensity of fluorescence in the absence of oxygen. IPO2 is the intensity at a given partial pressure of PO2. K is the quenching constant for a particular fluorophore used. The intensity of fluorescence will decrease in proportion to partial pressure of O2 level of the sample. “A biosensor is a specific type of chemical sensor consisting of a biological recognition element and a physiochemical transducer, often an electrochemical or an optical device. ”* the biological recognition element is capable of detecting the presence/concentration or activity of a certain metabolite in solution, in this case the biosensor will detect the biomarker, alkaline phosphatase (ALP). There are two ways for recognition to occur, by either a biocatalytic reaction where the biosensors are derived from enzymes, microorganisms, tissue slices, and biomimetic catalysts2, or a binding process.

The most important aspect of a biosensor is that it has a biological recognition site which can interact with alkaline phosphatase. The interactions of this biological site on the biosensor with the target analyte produces a signal which the transducer is responsible for converting in to an electrical signal which can be quantified. The mode of transduction can be based on optical spectroscopy or electrical transduction. Electrical transduction includes current potential, piezoelectric transduction, impedance etc. Optical transduction includes absorbance, fluorescence, luminescence, reflection. There has been various types of enzyme-based biosensors with optical detection developed for measuring metabolites such as cholesterol, ethanol, glucose, uric acid, glutamate, glutamine etc. Enzyme based biosensors are a very well-studied and mature topic. Enzyme-substrate interactions have very high specificity which results in a quick efficient process. Enzyme-based biosensors require the integration of a biocatalyst2. [image: ]The above picture shows the general scheme of a biosensing technique.

In this project optical detection method will be used to observe the cytoxic effect of Aloe Vera on Ht-29 colon cancer cell. Lengthy studies have been carried out in the area of sensors but in particular in optical biosensors. These studies have been vital for the development of medicine, environmental control and industry. This research and study needs to be continued for more developments in medicine and clinical practices. Biomarkers are relatively new and still a new medical approach. “The term biomarker in medicine most often stands for a protein measured in the circulation (blood) whose concentration indicates a normal or a pathological response of the organism, as well as a pharmacological response to the applied therapy. ”The advantage of biomarkers is that pathological changes can be identified early before the disease which would allow for the correct preventative measures to be taken. Or the pathological changes are identified early in the disease which means the right therapies and maintenance could be carried out to prevent the disease developing any more than it already has. This technique has been developed for the detection of prostate-specific antigen (PSA)4, this is measured in sera for early detection. PSA is produced in the prostate glands. The one problem with the detection of PSA is that PSA serum level is influenced by many factors. High PSA levels is not always caused by prostate cancer, a low PSA level cannot exclude malignant cells.

Therefore diagnosis can result in a false-positive or false-negative results. This shows that a very important characteristic of a biomarker is high specificity to save healthy patients being treated for a non-existent disease or ill patients not being treated for a disease. Proteins are a common biomarker used but DNA, RNA and other metabolic products may be used as biomarkers too. MicroRNAs are under investigation as a biomarker for the prognosis of cardiovascular diseases. ALP is the biomarker in this experiment. This enzyme has many different naturally occurring synthetic substrates. Mg2+, Co2+, and Mn2+ are all activators of this enzyme. PO43-, BO33- and CN- are all inhibitors of ALP activity. *A buffer which is used to maintain the pH may affect the activity of the enzyme. Buffers for ALP assay are classified as (1) inert, (2) inhibiting and (3) activating. *ALP is present all over the body; cell membranes and surfaces, bones, liver, placenta, small intestine, kidney. The exact metabolic of ALP is not yet understood but it does play crucial roles in the calcification of bones and the transport of lipid in the small intestines. ALP has been of particular interest in the measurement of bone disease and hepatobiliary(gall bladder, bile ducts or bile) disease. When levels of alkaline phosphatase are high ALP can dephosphorylate adenosine triphosphate (ATP) which then causes inflammation. Dephosphorisation is the removal of a phosphate (PO43-) group from ATP (an organic compound) by hydrolysis (ATP4− + H2O ⟶ ADP3− + HPO42− + H+). ATP acts as free energy in all living organisms.

The detection of biomarker ALP at Point of care testing (POCT) can detect early colon inflammation. POCT is defined as a laboratory testing that is carried on or close to the place the patient being tested is located5. POCT is a pathology services that uses bodily fluids such as blood, urine, and other metabolites to test certain parameters. The advantages of developing electrochemical sensing for point of care testing of the release of biomarkers from cells are numerous. One of the biggest problems the health care system in Ireland faces is the lack of beds for patients and being short staffed in hospitals and doctor surgeries. There is a greater demand compared to services provided. Spaces in hospitals are being taken up by people who are ill and awaiting tests such as blood results to come back which could take up to a week. This patient then takes up a bed and takes up time of the doctors and nurses. POCT allows nurses, pharmacists and even the patient themselves to monitor their diseases such as diabetes mellitus, hypertension, congestive heart failure and stroke prevention. The main advantage of POCT is that it delivers results very fast, this allows for the necessary treatment to start immediately for the patient. Recent data has shown that the diseases listed previously are all diseases that are the leading causes of hospital admissions in older patients. 5 POCT allows for real time monitoring where patients are in control of their own monitoring of their own disease.

An example includes diabetes where the patient monitors their blood sugar level. This is monitored by pricking one’s finger with a sharp needle called a lancet and placing a drop of blood on a test strip. Upon inserting that test strip in to a meter, a figure appears which displays the blood sugar level. This result is received in seconds and information can be stored for future use. POCT is the future of diagnostics and maintaining diseases for patients. It is continuously evolving in today’s medical industry. Chronic diseases are increasing rapidly in the United States of America and the aim is to not find a cure for these diseases but prevent development and onset of these disease and maintain the disease where the patient will continue to have a high standard of life while managing the disease themselves. The aim of this project is to attain a way of POCT for the enzyme alkaline phosphatase. High levels of alkaline phosphatase can dephosphorylate ATP that induce inflammasome, then diseases starting with colon diseases and ulcers which lead to colon cancer. “Alkaline phosphatase, enzyme that is normally present in high concentrations in growing bone and in bile. It is essential for the deposition of minerals in the bones and teeth. ”Colon cancers is one of the most prominent tumours among patients world-wide and is one of the big killers. There are ways to treat colon cancer such as chemotherapy and other target-oriented drugs. This POCT would allow to detect the release of biomarker, ALP before the cancer is developed which would allow treatment to prevent colon cancer. Colon cancer is the 3rd most common cancer in the world.

The larger fraction of cases have been linked to environmental causes rather than heritable diseases. In Europe there are 250,000 new cases each year. It has been seen that colon cancer in populations have increased with increasing industrialisation of areas. Colon cancer is associated with inflammatory bowel disease (IBD) which is hard to treat and results in many deaths each year. One of the main therapies to fight this illness has been the use of chemotherapy 5-FU drug but colon cancer cells have been becoming resistant to this drug and it is not understood why. In Ht-29 colon cancer lines it is seen that there are high levels of miR-21 present. It was investigated if these high levels had an effect on Ht-29 cells being resistant. It was seen that inhibition of the miR-21 cells led to hypersensitivity of the Ht-29 cells. This showed by targeting the miR-21 cells instead resistance to the 5-FU chemotherapy would be overcome. In this project tests will be carried out on the Ht-29 colon cancer lines. Colon disorders and ulcers lead to colorectal cancer. There has been a vast amount of supporting evidence and research in the last 10 years that has shown the connection between inflammation and tumorigenesis. Inflammation of the bowel is something that can develop in to colorectal cancer.

The molecular motions in which inflammation promotes cancer are still being discovered. Bowel disease is a high risk of developing to colorectal cancer. Inflammation is a response triggered by damage to living tissues. Inflammation can occur in different forms of sporadic. It can also be genetically inherited. The key is to detect when inflammation begins and manage it, while maintaining a high standard of living by preventing the development of colorectal cancer. Aloesin has displayed properties of anti-inflammatory properties. It has been used for many years as a home remedy. Aloesin has been known to be biologically active in colitis of rats which refers to the inflammation of the inner lining of the colon. Studies have shown that plant and food consumption are closely linked to many health benefits. Aloe Vera is used world-wide to help with the soothing and healing of burns by being applied directly to the skin. It is used for cosmetic uses to remove dead skin cells and leave skin feeling soft in moisturisers and lotions. However it is not clear exactly what component in Aloe Vera is related to which activity. The most complementary therapy and alternative medicine used for gastrointestinal disorders is herbal medicines.

Aloe anthraquinone and aloesin have been proven to both have strong inflammatory effects in murine macrophages11 which is the bone, marrow and peritoneal cavity. From these results it could be put forward as a hypothesis that aloin and aloesin, components of Aloe Vera could combat inflammation in the colon. The Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Republic of Korea carried out an experiment on rats to investigate the effects Aloe Vera components would have on anti-inflammatory activities in the colitis model.

Rats were split up in to different groups and their diets were all different with varying amount of Aloe Vera components. The results showed on day 21 the rats that were being fed aloe components had significantly lower disease activity index scores compared to the control group. The conclusion of the experiment was that dietary intake of aloe components improve inflammation response of DSS-induced (Dextran Sulphate Sodium) ulcerative colitis in rats. These Ht-29 cancer cells will be maintained by using cell culture techniques. These cancer cells are grown under controlled conditions generally in vitro.

After the cells of interest are obtained from a living organism they can be controlled and grown in a favourable environment of the correct nutrient supply, temperature and pH. Typical growth conditions for cells are 37 degrees Celsius, 5% CO2 and a relative humidity of 95%. Some cells require extracellular matrix, a special surface to attach to and grow more efficiently. The cells will be supplied with a media which will contain a carbon source of energy such as glucose, amino acids and vitamins to help with growth. A culture lab is required for the growing of cell culture. It is necessary to carry out aseptic techniques when interacting with cell culture.

Where actions are carried out under sterile conditions and free from any contamination. The cell’s viability will be monitored using fluorescence measurement. Fluorescence is a form of luminescence where light is emitted by a substance that has absorbed light or other electromagnetic radiation. Fluorescence happens by a process illustrated by Jablonski diagrams. Electrons become excited by the absorption of light energy and move from the ground state energy to an excited state. Electrons get to the lowest energy level of the excited state via internal conversion. These molecules are then capable of fluorescing. When demote to the ground state they release light which is fluorescence.