Universidade Tcnica de Lisboa Instituto Superior Tcnico InescMN

Detection of Pca biomarkers using microfluidic system integrated with detection

Supervisors: Professor Joo Pedro Conde Dr. Narayanan Srinivasan

3rd February 2014 | Miguel Reis

Detection of PCa biomarkers using microfluidics systems with detection

Detection of PCa biomarkers using microfluidics systems with detection

Abstract

Detection of PCa biomarkers using microfluidics systems with detection

Index

Abstract................................................................................................................................ 3 Index..................................................................................................................................... 4 Introduction.......................................................................................................................... 5 PoC analysis Clinical Diagnosis..6 LoC..7 PCa ..8 PCa biomarkers..9 PSA model10 ELISA.11 ELISA methodologies...12 ELISA assays...12 Competitive.13 Sandwich14 Detection.14 Disadvantages..15 Microfluidics Advantages Principles, physics Applications/modifications Microfabrication (Engineering) Control of surface chemistry. PDMS.. Objective of Thesis

Detection of PCa biomarkers using microfluidics systems with detection Approach Reversibility ???

Introduction

Detection of PCa biomarkers using microfluidics systems with detection

Point of Care (PoC) analysis Clinical Diagnosis

Point of care analysis is presented now-a-days as the most valuable method for patient testing due to the advantages it conveys over the standard laboratory testing. PoC testing has already gain a considerable percentage of the in vitro diagnostics market however this is due to the large diabetes monitoring chains in it. Outside the diabetes market the use of PoC devices is a bit more controversial partially due to sensitivity and reliability issues.(B, K)The main concept behind POC is the have the patient tested in the spot returning the results instantly which allows an immediate course of action to be taken by the diagnostic entity. This brings up many doubts about the quality and reliability of the results presented by such test. (A, B) This, alongside with integration present the current challenges of PoC devices development. Fluid handling tasks such as fluid delivery, mixing, separation and concentration are simple tasks to handle in a laboratory which need to be integrated into such device. The aim is to develop a fully integrated and automated miniaturized system able to perform samples analyses with high sensitivity and accuracy on a single portable platform.(C,D) In the last few years microfluidic platforms have demonstrated to be efficient in integrating different modules for sample processing leading towards a fully automated analysis system. Microfluidics is also presents other useful characteristics for developing PoC devices such as reduced costs and high sensitivity as well as accuracy. (A, C) Also digital microfluidics, multiphase flow systems and multielectrode arrays are a few other platforms that may provide the tools needed for PoC diagnostics. (C,E) Digital Microfluidics (DMF) for instance is an interesting technology since it eliminates the need for external pneumatic controls and valves in order to control reagent and sample flows. DMF concept is to manipulate sample volumes the size of a droplet. These droplets are driven over a micropatterned plate which uses an electrowetting mechanism based on the surface energy changes due to applied potencial. This allows for different steps to be programmed and executed orderly making the manipulation sequence similar to the bench-top procedure using smaller volumes, higher automation and considerably less time.(I, F)
Figure 1 (A) Microelectrode array device (adapted from F); (B) Digital microfluidic device (Adapted from G); (C) microfluidic platform (adapted from H)

Detection of PCa biomarkers using microfluidics systems with detection

Lab-on-chip (LoC)

In light of all these new technology comes forward a new concept named Lab-on-chip (LoC). In this concept is embedded the idea of developing a miniaturized laboratory on a chip which can perform complex top-bench tasks, reducing reagents waste and the time of the analysis itself. Such devices have the size of millimeters to few square centimeters in size. LoCs belong to a subset category of MEMS (Microelectromechanical systems) and are often based on microfluidic technology in such a way the physicals principals behind such device are withdrawn from the microfluidics field. All this is manageable on a microscale due to the difference in behavior of fluids at this scale. The manipulation of amazingly small volumes (nano to pico liters) also allows for a more accurate control of the concentrations and interactions between liquids. (J) Lab on a chip technology can be applied to a wide variety of clinical analysis. The most common is in the measurement of blood sugar levels by diabetic patients. This technology may also be used for the detection of biomarkers for liver failure, kidney damage and CVDs (cardiovascular diseases). Not only for biomarkers but also for the detection of virus, LoCs technology may bring forward great progress. LoC is particularly useful for these essays since it provides a platform for miniaturization of standard ELISA analysis used for biomarkers and viral clinical analyses. (K, L, M, R) Another interesting use of LoC devices is in the miniaturization of PCR assays. The application of these devices for quantitative PCR and Real-time PCR is another aspect demonstrating the versatility of the LoC concept. (N) Another interesting parameter to look at is the amount of different technologies than may be performed in this device. PCR, as we just saw is one of these technologies alongside with flowcitometry, microarrays, aqueous two-phase systems and microfluidics.(N) Some detection methods are also applied to such devices. This includes labeled antibodies with HRP-enzyme (chemiluminescence), fluorophores (fluorescence), which light emission can be detected through the use of a microscope or in a much more integrated and simplistic manner, through the use of photodiodes. (O) Overall the LoC devices provide innumerous advantages in comparison with most of the current methods used. Such advantages include: reduction of reagent and sample volumes; faster analysis (faster reactions inside the device mostly due to the size and scaling effect); easier control of various environmental parameters such as concentration, temperature and chemical reactions time; portability; automation; Higher throughputs; lower fabrication costs. However some disadvantages also rise, these being: the experimental conditions may not always scale down in a positive manner leading to further complications; signal-to-noise ratios; lack of accuracy in Microfabrication in comparison with bigger scale engineering. (P,Q)

Detection of PCa biomarkers using microfluidics systems with detection

PCa (Prostate Cancer)

Prostate cancer is now-a-days one of the leading causes of cancer related deaths in men, particularly in the western countries, being the most prevalent form of sickness in Europe. WHO statistics register about 200,000 new cases of prostate cancer every year. The prostate tumor grows penetrating peripheral tissues (perineural invasion) such as lymphatic channels and nodes. The lymphatic nodes are the most likely tissue to host the first metastases (T, S). After the metastases spread into the blood stream, one of the first targets are the bone structure, forming osteocleorotic metastases. (U)

PCa biomarkers

A significant variety of biomarkers are known for PCa, although each one with different characteristics and different reliabilities, presenting different pieces of information about PCa. From proteins to mRNA and also metabolites concentrations such as calcium may be measured for obtaining information on tumor development. PSCA (prostate stem cell antigen), for instance, is a GPI-anchored cell surface protein found in blood that depicts with a reasonable degree of accuracy the progression of PCa (PSCA-1). High expression of this protein is observed in about 90% of prostate cancer patients. As for PSCA-2 it is known to have a Jekyll and Hyde molecule behavior and is known to be presented in high values in cases of bladder and pancreatic cancer. (Y, AA) PSMA (prostate specific membrane antigen) is another PCa biomarker found in tissue, serum and cells. PSMA is a zinc metalloenzyme residing in membranes facing the extracellular space. It is thought to be one of the most upregulated proteins in prostate tissue, going up has far as 12-fold when compared to noncancerous patients. However it has been reported increase expression also in other tissues such as the brain, liver and kidneys, therefore requiring calibration strategies when taking into account PSMA concentration in a clinical analysis.(Z) PAP (Prostatic acid phosphatase) is another serum enzyme which upregulation carries indication of prostate cancer although it is not a very sensitive result. A variation in Calcium concentration can is also known to be an indicator of a disturbance in the homeostasis. A direct correlation between the increase in calcium concentration and the development of a prostate tumor may be established. These changes in the concentration are due to the change in the expression of a calcium sensing receptor (CaSR) with affects the calcium intake. (BB) Many others biomarker possess relevance in the diagnosis and prognosis of PCa. Some of these are EPCA (Early prostate cancer antigen), GOLPH2 (Golgi phosphoprotein 2) and even sarcosine. (CC) Nevertheless a consensus on values for diagnostic purposes does not exist yet among the scientific community on these biomarkers. There is one though which has awakened some interested in the last few years. This is prostate-specific antigen.

Detection of PCa biomarkers using microfluidics systems with detection

Prostate-specific Antigen One of the most prominent PCa biomarkers is PSA (Prostate-specific antigen), used particularly for early diagnosis and also for monitoring the progression of the tumor. PSA, also known as kallikrein-3 (KLK3) is a grycoprotein enzyme from the kallikrein-related peptidase family, secreted by the epithelial cells of the prostate gland. PSA may allow the detection of PCa as much as 5 years before symptoms start. Although this molecule is intended to dissolve cervical mucus facilitating the sperm entrance in the uterus, small quantities of it roam in the circulatory serum. Since this protein has a proteolitic function, the biggest percentage in the serum is a complexed form of PSA bound to another protein, most commonly 1antichymotrypsin (PSA-ACT) or 2-macroglobulin (PSA-A2M). This is because of the proteolitic activity of this protein requiring a silencing protein to be coupled to PSA in order to inhibit its activity. Nevertheless, a non complexed form of PSA exists in the blood current in a small percentage being designated free PSA (fPSA). The ratio between the two different forms is the key for correlating with a possible PCa or BPH (Benign Prostatic Hyperplasia) diagnosis. Also the ratio may change in a significant way just so that information concerning the evolution of a tumor may be extrapolated. In healthy man PSA is present in very small amounts in the serum whereas in cause of prostate disorders the values are more elevates. Since naturaly every man has different basal PSA values, some calibration of these values require calibration in order for the reading values to be relevant. This is where the ratio between the fPSA and PSA-ACT come in. The total amount of PSA in the serum (both fPSA and PSA-ACT) is designated by tPSA(total PSA). (S, W, T)

PSA antibodies The use of PSA antibodies in ELISA testing are the standard test in clinical laboratories, therefore it becomes important to define the quality and importance of the antibodies chosen. For fPSA and PSA-ACT, anti-fPSA and anti-PSA-ACT antibodies respectively are used for measuring these molecules concentrations. In terms of measuring the overall amount of both forms of PSA a standard anti-tPSA antibody has proven to be inefficient by presenting changes in the affinity for the two different forms of PSA. It would happen that anti-tPSA antibody would have more affinity to the complexed form since it is present is a higher concentration. This led to the development of equimolar total PSA (emt-PSA). This antibody maintains the same affinity for fPSA and PSA-ACT independently of the concentration of each one avoid an over or underestimation. This way PSA ratio can now be measured more efficiently. (V) Using the emt-PSA antibody we have a capture system for two different molecules (the fPSA and PSA-ACT) which affinity is not affected by the targets concentration therefore allowing for a correct measurement of both a single essay.

Detection of PCa biomarkers using microfluidics systems with detection

ELISA
The enzyme-linked immunosorbent assay (ELISA) is a technique which makes use of antibodies to detect the presence of target molecules in different types of samples. This technique uses the principals of immunology to capture the molecules of interest. Signal detection in ELISA is usually done through some form of quantification of light emission from the reaction mixture. ELISA is now a days the standard diagnostic tool in terms of metabolite quantification in liquid samples. This also makes ELISA a good quality-control check for further tools developed by the medical research industry.

Different ELISA methodologies Two different strategies are used when planning an ELISA test. An ELISA signal comes from a source of light coupled to the detector antibody. This detector antibody may be a primary antibody or secondary antibody depending on the chains of events of the ELISA strategy. In a direct ELISA the detector antibody (the antibody coupled with a light emitting enzyme) is linked directly to the antigen (target molecule).This usually only allows for one antibody to link to the antigen which may translate into a weak signal depending on the concentration of the antigen. There is however another strategy called indirect ELISA in which the enzyme-coupled antibody is not specific for the antigen of interest. These antibodies are called secondary antibodies and their specificity is to mouse antibodies. In this strategy the primary antibody (specific to the antigen) is not coupled to any enzyme and simply binds to the antigen. After this first incubation step the secondary antibody is added to the mixture and binds to the primary antibody. This allows for the binding of more than one secondary antibody to the primary antibody therefore increasing the signal. The indirect assay carries some advantages over the direct approach, these being the bigger number of binding sites for the coupled antibody (which amplifies the signal), and the availability of an enzyme-coupled antibody for assays with different antigens, since the secondary antibody binds to whichever mouse cloned antibody that may be used.

Figure 2 (a) Direct ELISA, with antigen coated on surface (purple) and enzyme-coupled (yellow) antibody (orange) bound on top; (b) Indirect ELISA, with antigen coated on surface(purple), specific non-conjugated antibody (orange) bound to the antigen and enzyme-coupled (yellow) antibody (red) bound to the specific antibody.

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Detection of PCa biomarkers using microfluidics systems with detection

ELISA assays

Sandwich ELISA is one the most common ways to used ELISA. In this methodology, a surface is coated with a capture antibody for the antigen of interest. Then, the remaining free surface is block usually with BSA (bovine serum albumin) or SSDNA (salmon sperm DNA) and only after this step, the testing sample is run on this functionalized surface. Finally the detection antibody (an enzyme-coupled antibody specific for a different epitope of the antibody) is added, binding to the antigen already trapped in the primary antibody. A washing step is always required after each incubation step. The signal measured in the detection step is directly proportional to the concentration of the antigen in the tested sample. A different way to do an ELISA is by performing a competitive assay. The strategy for measuring concentration is different in both. Unlike the sandwich assays, in a competitive assay, the signal read in the detection step is inversely proportional to the concentration of the antigen. In the assays the surface is coated with a solution of the antigen followed by a blocking step. After this a previous mixture of a known concentration of enzyme-coupled antibody and the testing sampled is ran in the surface functionalized with the antigen, and the remaining free antibody in the mixed solution will bind to this surface bound antigen, whilst the antibodies which bound to the free antigen will be washed away. This was the bigger the antigen concentration in the sample, the fewer antibody molecules will be left free to bind to the surface bound antigen, depicting an inversely correlation.

Detection

For ELISA, light emitting enzymes used are coupled to antibodies in order to detect this light for quantification. This includes fluorescence, chemiluminescence and colorimetric assays, which require the use of appropriate enzyme labels and the corresponding substrate for the detection system. Horseradish peroxidase (HRP) is one of the most commonly used enzyme and uses luminol as substrate for the production of a light emitting product. Other substrates may be used with HRP, such as TMB (3,3,5,5-tetramethylbenzidine) and also other enzymes such as alkaline phosphatase (AP) can be used as a detection system instead of HRP. Recently the use of QDs (quantum dots) conjugated antibody, instead of enzyme-coupled antibodies in a standard ELISA protocol, has brought a redefinition of this assay. However this variation in the detection system is just a different detection system applied over a systematic protocol, ending up in similar results. Quantum dots are nanocrystals capable of emitting light in different wavelength depending on its size. These are a new approach to the detection systems integrated in ELISA. (DD, EE)

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Detection of PCa biomarkers using microfluidics systems with detection

Disadvantages

Either detection system has its own strengths and weaknesses. HRP for instance is less expensive than alkaline phosphatase (AP), although it is also incompatible with sodium azide, a common preservative present in antibodies commercial buffer, used for prevent microbial contamination. Furthermore the presence of metals in solution is also known to affect peroxidases activity. AP has its own issues as well. Much like HRP, alkaline phosphatase is also inactivated by some conditions and metabolites. Chelating agents, acidic pH and inorganic phosphates are some of the factors which affect AP functioning. (DD)

As it is clear, drawbacks are present in different steps of any ELISA protocol and these drawbacks have been the driving force to the optimization of this technique, coming up with new approaches to overcome these disadvantages. One of the most concerning disadvantages of the ELISA, is the amount of wasted reagent per run, and the considerable amount of time necessary in comparison with some more recent methods. More recently, new approaches have emerged, to define a whole concept of the ELISA, and optimize this technique in order to make it more rentable. Microfluidics is the new approach which is redefining the ELISA assay making it more inexpensive and less time consuming.

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Detection of PCa biomarkers using microfluidics systems with detection

Microfluidics
Microfluidics is a multidisciplinary field of engineering science which integrates biology, nanotechnology, chemistry and biotechnology into one field for development of a working miniaturized platform. Microfluidics brings forward the concept of miniaturization and integration, optimized in a way to make the best of the scale reduction advantages. Improvement in the behavior, control precision and manipulation of small volumes are the perks of this approach making a bold statement in the sample analysis industry. Some of these advantages go hand-in-hand with the methodologies defined for LoC devices and it is therefore the base of development of such devices. Other advantages of this system are small sample and reagent consumption, reduced size of the overall apparatus, low energy consumption and the difference in the physical behavior of solution at a sub-millimeter scale. The fact that a microfuidic device handles samples in miniaturized scale brings forth the differences in behavior of these samples, but also other advantages which the microscale superimposes on the macroscale devices besides the benefits already mentioned in the Loc section. High throughput screening is one of the benefits of using Microfluidics platforms to perform chemical or biological reactions. This is because Microfluidics provides the integration of all sample handling steps into one small area of device, allowing for the parallel duplication of the same steps, delivering the greater number of results with in less time and with less reagents consumption. The easiness on the manipulation of the devices design and Microfabrication is also one of the great advantages. The need to experiment with different designs in science research may sometimes delay the progress of said research. Microfluidics devices are mostly fabricated through the use of molds, which carry the design of the device and stamp this design onto such polymers as PDMS (polydimethylsiloxane). This way the manipulation of the design is solely dependent on the time consumed for the fabrication of a new mold, and this same mold maybe be used several times to stamp silicon based polymers.

Microfluidic platforms are devices for integrating various fluid controlling tasks such as monitoring, transport, mixing, chemical or biological reaction, incubation and detection.

Microfluidics Advantages Principles, physics

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Detection of PCa biomarkers using microfluidics systems with detection Applications/modifications Microfabrication (Engineering) Control of surface chemistry. PDMS.. Objective of Thesis

References

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Detection of PCa biomarkers using microfluidics systems with detection

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