Prostate Cancer is amongst the common types of cancer in US, Europe, UK and even globally, and the second most main origin of mortality after lung cancer in men. Usually, PCa is sensed in older men of age, with a good number of cases identified in men who are middle aged, normally between the age of 40 and 50 years. This type of cancer is asymptomatic and slow growing; therefore, detecting it is a challenging issue amongst clinicians. Clinical symptoms are the same as those of prostatitis, which makes it a challenge in accurately distinguishing between the two. Due to untimely and ineffective diagnostics, the PCa prognosis is poor generally, which is as a result of the high risk of metastatic development. Diagnosing PCa in time can minimize rates of mortalities and provide the opportunity for proper medical interventions. Thus, developing diagnostics that is reliable at early PCa stages is of high significance.
Currently, PCa diagnostics are basically done on detecting and quantifying the sum of serum prostate-specific blood antigen then through imaging studies and digitally rectal researches in case there is an observation of PCa. The introduction of the PSA test was done in the late 1980s and resulted to a great advance in PCadiagnostics. Though, limitations on its application are associated to its specificity absence since high PSA concentrations might not be related always to cancer. Elecvated levels of PSA can be credited to a number of conditions of benign such as infection and benign prostatic hyperplasia. In addition, development of tumours may appear before PSAs concentrations increases. False tests of PSA which are positive have resulted to overdiagnosis and finally overtreatment, which is applied as the gold diagnosis technique in clinical works (Kim, 2021).
A biological sensor defines a device that is able to detect an analyte through integration of biological element by using a transducer. The biomolecule is able to identify the analyte, and the changes are changed to an electrical signal by the use of a transducer. Electrochemical biosensor function through the conversion of chemical signals itno electrical signals. They are able to detect a number of biomolecules like blood ketones, haemoglobin, DNA, lactate, uric acid, cholesterol, glucose and many more. Thus, they can be applied in treating diseases which result from biomolecules imbalance. Though, they are applied mainly for biosensor applications and research on biosensors and where applications of delivery of drugs are scarce. As part of this project, there will be an application of a biorecptoraptamer which is a novel kind of and an electrochemical transducer. The application of the two will be used in measuring data that can be used on other applications, like calculation of risk of prostate cancer among patients (Cosnier, 2017).
The figure below shows the basic concepts of a biosensor.
This project aims at providing a platform for developing a simple, reliable and inexpensive diagnostics for prostate cancer. The objectives in this project include:
Conducting literature review on electrochemical biosensors and electrochemical apta-sensors to be specific
To carry out a number of IS and CV measurements on electrodes which have immobilized aptamers binding target molecules PCA3 in various concentrations (Afsarimanesh, 2019).
The CG-based aptamer of 277 bases specification was functionalized. The nucleotides sequence is known for binding a 277 aptamer section schematically as shown in the figure below:
Figure1, showing the bind of the CG 3 RNA –aptamer (the line and the red lines) to a PCA3 subsection including a 277 IncRNA bases transcripts (the line and the blue lines (Kumar, 2018)).
The immobilization of aptamers was done on the gold-screen electrodes printed on the surface through following the method and procedure schematically outlined in figure 2. The solution of the apatamer stock in a was diluted to 1 μM using HEPES binding buffer of 7.2-7.6 ph accompanied using 3mm of mgcl2 and 1 mm of 1.4 dithiothretiol. DTT is applied in breaking the bridges disulfide in the middle of two aptamers, releasing afterward the end gropus of a 3 thiol. The free thiol groups give allowance for the covalent aptamer binding to the screen of electrodes gold-printed on their surface. Before immobilizing is done the liquid samples of aptamer were activated through heating rapidly for one minute up to 95 degrees Celsius, followed by a one minute cooling at four degrees Celsius in a thermocycles (Costa & Miertus, 2015). Immobilization was conducted through casting the solutions of the aptamer onto the screen surface of the electrodes that is golden printed. After that, the incubation of the samples was done for 4 hours in a humidity chamber at a room temperature.
Aptamers that were not reacting were separated from the surface of the electrode through numerously rinsing the electrode with a non-folding buffer. The electrodes that were printed on the screen in gold with aptamers which were immobile were reserved at 4 degrees Celsius in HBB to ensure the aptamers did not coil. PCA3 solutions resuspended in phosphate buffer saline of PH 7-2 to 7.4 at 1000 ng/ml concentrations down to 0.1 ng/ml were applied in the measurements of the electrochemical (Carneiro & Leonardo, 2018).
Figure 2, showing a step by step aptasensor fabrication: a. purchase of aptamers; b. splitting of aptamers in HEPES binding buffer with an inclusion of Mgl2 and DTT; c. aptamers that are reactivated following thermcycling; d. immobilization of aptamers on Au electrodes with the labeling of the 50 redox done from its external; e. conformed changes of the configuration of the aptamer following the binding of PCA3 and leading in a subsequent rise of transfer of charge (Moretto & Kalcher, 2016).
The application of the assembled three-electrode gold screen with reference of electrode of Ag/AgCL were applied in the measurements of the cyclic voltammograms with the application of a Metrohmpotentiostat STAT8000P and DropSens, voltage ranging from -0.5 to 0.5V. This was applied in the measurements of CV with a 10mv step and a 10 mv/s scan rate. The recording of the CV cycles was done 3 times until there was an establishment of the current readings (Sun, 2019). Besides the cyclic voltammetry, the cathodic current time dependencies at –0.2 V on electrodes were recorded in the exposure of the PCA3 of various concentrations. Measurements of the electrochemical impedance spectroscopy (EIS) were conducted on inter-digitated dropsens electrodes printed on the gold screen surface with 50 fringes having a 5 µm spacing through the application of 4000 A EG & G impedance analyzer. The 50 Mv amplitude AC signal with no DC off-set which has the frequency variation from 0.1 Hz to 1 MHz was applied in these measurements. Both measurements of EIS and CV were conducted on electrodes that had immobilized aptamers absorbed in PBS ph 7.2 to 7.4 with various PCA3 concentrations ranging from 0.1 ng/ml to 1000ng/ml. Buffer measurements alone were applied as a reference. Also carried out were the negative control measurements through the application of bovine serum albumin of a concentration of 100 ng/ml on PBS (Luo, 2014).
As it has already been noted, prostate cancer is one of the most commonly known cancer types in Europe, UK, US and even globally and the second-most mortality cause after lung cancer amongst men. Usually, PCa is detected in men who are of old age, with a good number of cases being witnessed in middle aged men who are between the age of 40 to 50 years. This type of cancer is asymptomatic and slow-growing; therefore, detecting it in its early stages is a big challenge to clinicians. Clinical symptoms are the same as the benign prostatitis, which makes it a challenge in distinguishing accurately between the two. Due to lack of proper and in-time diagnostics, the PCa prognosis is poorly generally, which is as a result of the high risk of metastatic development. Diagnosing PCa early enough can minimize the rates of mortalities and increase proper medical intervention opportunities. Thus, developing of diagnostics that is reliable at the early PCa stages can be of great importance (Rafat, 2017).
Current PCa diagnostics are based on detecting and quantifying the total serum prostate-specific antigen in blood then in case of suspecting PCa, digital rectal research and current researches. The introduction of the test of PSA was done in the late 1980sand resulted to a great improvement in the diagnosis of PCa. Though, limitations in its application are associated to it lacking specificity since high PSA concentrations might not be related to cancer always. Elevated levels of PSA can be credited also to a number of benign conditions like benign infection or prostatic hyperplasia. In addition, the development of tumours may happen before the PSA concentration increases. PSA tests that are false but positive have resulted to both over-diagnosis and finally over-treatment of men in huge populations who go through prostate biopsies that is needless, that is applied as the method of gold standard diagnosis in clinical research. Therefore, identification of alternative biomarkers that is specific to prostate cancer and development of techniques for detecting them in early stage of the disease in needed (Zhang & Wang, 2017).
A wide range of PCa biomarkers that is promising like PSMA11, TMPRSS2, ERG gene fusion, PCGEM 1 and RNA urine biomarker(DD3PCA3) have been known all to be over-expressed as far as prostate turmours is concerned. The diffential display code 3 (DD3PCA3) gene, also referred to as prostate cancer antigen 3 (PCA3) was established in the year 1999 and currently it is one of the particular malignant PCa makers. The levels of PCA3 can predict the outcome of the prostatic biopsies, particularly in combining them with other biomarkers of PCa like PCa can minimize the possibility of false-positive results. PCA3 can be detected in blood, collected urine after DRE and standards tests of urine, these PCA3 features make it the best biomarker for early diagnosis of PCa that is invasive (Yogeswaran, 2016).
Typically, PCA3 detection has been carried out using the amplification of RT-Qpcr. Currently, detecting the PCA3 in the samples of post-DRE urine is part of the progensa test commercially established and accepted for clinical uses in the USA. The basis of the progensa assay on detection of both PCA3 and PSA markers through the application of measurable nucleic acid amplification with high specificity and sensitivity. Though, the test is expensive and time-inefficient. The PCA3 biosensors development for cost-effective, express and accurate PCa diagnostics is a subject of great significance. Enough progress in development of biosensors for diagnosis of cancer with the inclusion of prostate cancer has been so far made. A current electrochemical biosensing advancement which includes nano-carbon materials such as graphene oxide, graphene, cabon nanotubes, gives allowance to a substantial improvement in discovery and sensitivity. A detailed review of the current uses of nano-carbon materials in biosensing showed the detection of a number of cancer biomarkers in reduced concentrations to the level of fm (Singh, 2018). PSA electrochemical detection using a LOD of 13 pg/ml has been testified. Though, the PCA3 finding was not mentioned in this research. Electrochemical biosensors are specifically attractive for biomedical uses since they have got a combination that is unique low cost, simplicity in application and high sensitivity.
The PCA3 optical detection at concentrations that are between 200 fm and 5nm through the use of nanoparticles of graphene-oxide adapted with reduced oligonucleotides particular to PCA3 sections has been reported. Though, the assessment of that specificity was not done. Both optical and electrochemical detection of a short PCA3 ssDNA succession which are similar to the real IncRNA PCA3 sequence was attempted recently. The limits of detection reported for impedance spectroscopy were 83 pm, 0.9 nm for UV-vis absorption spectroscopy and 2nm for cyclic voltammograms. Though, the IncRNA PCA3 detection was only attempted qualitatively. The sensors could differentiate between extracted PCA3 from various cell lines which have a high, negligible and low PCA3 concentrations (Ozsoz, 2017).
The focus in this research has been emphasized on the further electrochemical sensors development for the PCa biomarkers detection. One particular challenge of electrochemical biosensing, that is, the necessity for redox chemicals in the solution tested can be determined through the application of a redox-labelled apmaters as bioreceptors. Aptemers are not natural, averagely short RNA or DNA-based constructs which have a specific sequence of nucleotides designed to stick to particular targets from small inorganic and organic molecules to molecules that are large in size like proteins. The mixture of high stability and selectivity with reduced synthesis cost and easily modified with different functional groups enables aptamers to look attractive for several applications (Pumera, 2017). For instance, labeling aptamers which have redox groups that are active electromechally enables them to be specifically good-looking for electrochemical biosensing. The technique is grounded on the tributary structural changes of the aptamer once it has abided to the molecule targeted. Conformational changes ensure that the redox label is brought closer towards the electrode which leads to successful increase in the transfer of charge. This tactic has been adapted effectively for mycotoxins electrochemical detection, specifically ochratoxin in investigation of food, and it has been used in detecting heavy metal ions in water and equally detection of in-vitro of dopamine (Alegret & Merkoci, 2017).
In this case, a high affinity novel of CG-3 RNA-based aptamer particular to 277 PCA transcript bases will be applied. The labeling of the aptamer is done with forrocene which allows the 277 nt fragment detection from the PCA3. The interesting results are for basic science because the outcomes which result from changes in minor structures of the two including the aptamer and PCA3 during the outcomes are unknown. The observation in this study is a step that has been made towards achieving the long-term motive of developing an accurate, novel, cost effective and simple diagnostic prostate cancer tool (Rinken, 2015).
Typical recorded CVs in electrodes which had immovable aptamers exposed to various PCA3 concentrations are explained in figure shown in 3a. Distinguishing peaks of anodic current (at around 0.15–0.2 V) and cathodic current (at around 0.25–0.3 V) can be related with the redox activity on the label of the forrecene aptamer. These results are the same as those of the CVs that were observed in the early research comprising aptamers labeled with ferrocene. Furthermore, aptamers that were not labeled did not indicate that electrochemical behavior. The amplitudes of both currents of cathodes and anodes can be seen to be going up with the rise of PCA3 concentrations as shown I figure ).
Figure 3a, showing a typically set of cyclic voltammograms(CV) for various PCA3 concentrations. Figure 3b shows a concentration reliability on the changes on the currents of the cathodes and anodes and also LOD evaluation.
In figure 2b, there is a shown dependency of average changes of both currents of cathodes and anodes on the concentration of the PCA3, that can be applied as calibration curves. For zero concentration the CV curve of the PCA3 was applied as the reference. The noise level in the measurements of the CV was approximated to be 5 percent of the level of the signal. The increase observed in the current that was correlating with the concentration of PCA3 increase can be defined by the charge transfer improvement between the labels of ferrocene and the electrode as a result of the changes in the secondary structure of the aptamer on binding to the 277 nt fragment of lncRNA PCA3 (Pujadó, 2019). The process is shown in figure 2e. As it can be seen, there is no linearity in the calibration curves, and most probably, they symbolize the lower part of the standard sigmoid curve. Within the concentration range applied in this case, saturation was not attained. The detection limit can be observed to be lower than the lowermost concentration of the PCA3, at 0.1 g/ml applied in the tests.
The values of the LOD were approximated through linear extrapolation of the curves of calibration to be thrice the level of noise, for example 0.015 in average changes. It can be observed that the recent measurements of the cathode were seen to be a bit sensitive (LOD ≈ 0.04 ng/mL) in comparison to the current of the anode, (LOD ≈ 0.08 ng/mL). in the two cases, there is a very high sensitivity and impending levels of ppt. The high sensitivity is attained here through detection of PCA3 which is the same as the sensitivities of other earlier reported electrochemical aptasensors. Negative controls test on binding BSA to anti-PCA3 aptamers were conducted but there was no response shown (Bollella & Katz, 2017).
Typical electrochemical impedance spectroscopy results are presented in the figure4a as reliabilties of the total value of the invented part impendence against the real part, which is referred as Nyguist plots. The points of data in every curve correspond to various AC signal frequencies, the arrows showing the increase of frequency from 0.1 Hz to 1 MHz. the almost real observed semi-circular pattern is real for systems without limiting diffusion in receptor analyte interactions, which have been used in the samples including a monolayer aptamers which are immobilized on the gold electrodes surface. Thus, the simplified same circuit model with no diffusion impedance is shown as a figure 4a inset. This can be applied in modeling the measurements of EIS results. The Z impedance of the simplified equivalent circuit circulation is shown below (Bollella & Katz, 2017):
In the above circulation, Z0 and Z00 are real and imaginary impedance parts respectively. RDL and CDL are the resistance and capacitance of a two layered electrode surface respectively and RS is the solution of the bulk resistance. At minimum frequencies,
Thus, the featured interest parameter, for example, a double layer resistance RDL, can be worked out without calculating the data of EIS fitting as follows:
In practice, both Z0 and Z00 represents the real z values at lowest, which is at 0.1 Hz and the highest, which is at 1 MHz frequencies in that order. The RDL dependence on the concentration of the PCA3 is shown in figure 4b (Cosnier, 2017). The reduction in RDL with the rise in the concentration of PCA3 relates well with the current rise of the DC in the measuremnts of the CV and approves the electrochemical apta-sensing concept shown in figure 2e. A significant reduction in RDL in the measurements of EIS which resulted from the binding of PCA3 aptamer compared with somehow little changes on both currents of the anode and the cathode in the measurements of the CV resulted to the decision that the measurements of the EIS are more profound in comparison to the measurements of the CV. The reduced limit of detecting the EIS technique could be approximated through plotting 1 /RDL against the PCA3 concentration. The graph is shown in figure 4b as an insert. The measurements of EIS gave allowance to evaluating RDL with an accurateness of around 1 ?. Applying the RDL ≈ 1 k ? value at zero PCA3 concentration as a locus, The level of noise can be approximated to be ?(1/RDL) = ?RDL R 2 DL = 10−5 (S) or 0.01 mS, which as at zero level practically. The linear estimation intercept of 1/RDL vs. CPCA3 graph at minimum concentration results to the value of LOD at 0.03 ng/ml. The data in this case shows that the EIS technique is more sensitive in comparison to the CV technique (Alegret & Merkoci, 2017).
Figure 4a, showing a typically set Nyquist plots for various PCA3 concentrations. Figure 4b shows concentration dependence of the resistance of a double layer. Inset demonstrates the LOD evaluation.
The kinetics of binding PCA3 lncRNA to the aptamer layer on the surface of the gold electrode was examined by recording time dependencies of cathodic current at a −0.2 V fixed potential over various aptamer concentrations. Typical time dependencies of absolute values of changes in cathodic current |?Ic| = Ic − I re f c for various PCA3 concentrations are demonstrated in figure 5 (Moretto & Kalcher, 2016). The fitting of the data recorded to the increasing exponential function gave allowance to the time constant (τ) evaluation at diverse PCA3 concentrations. These data were applied in the further analysis of quantifying the aptamer affinity- binding of the PCA3.
Figure 5, showing a typical kinetics PCA3 binding of various concentrations to the aptamer. Inset shows the aptamer evaluation of the binding affinity of the PCA3.
A different equation describes the PCA3 aptamer binding for absorption, that is good in the case here, of analyte molecules adsorption on a monolayer of binding centers (Cosnier, 2017):
The first term of the equation above defines the adsorption of analyte molecules of concentration C[M] on the sites of binding available with the N-N concentration, where N defines the sites of binding concentration on the surface and the adsorption rate is ka mol-1 s-1, whereas the second term in the above equation defines the desorption of the molecules of analyte which is equal to the adsorbed molecules concentration (n) and the desorption rate is kd- s-1. Another estimation applied in the analysis of binding kinetics is the application of a single binding site assumption tto the process of binding that is comlex in between the PCA3 and the aptamer, which includes interactions between a numbers of nucleotides. Though, the approach proposed has been applied successfully in the same complex antibody-antigen systems of interactions, and in the previous work on aptamer-based biosensors. The above equation’s solution is a rising exponential function which is (Kim, 2021):
The values of the desorption and adsorption rates kd and ka) can be gotten as the intercept and gradient of 1/τ (C) dependence. Therefore, the association constant can be worked out as KA molar−1 = ka/kd, whereas the affinity constant KD[M] = 1/KA = kd/ka. Calculations of this kind were carried out for the kinetics curves in figure number 5 and the linear plot of 1/τ vs. C is given as an inset in together with the parameters evaluated. The resulting association values and the affinity binding constant for the PCA3 to the aptamer KA≈ 2.5·109 M−1 and KD ≈ 4·10−10 M, respectively, indicating on this reaction a very high specificity. The binding strength means that practically, it is irreversible because the PCA3 binding probability is 2.5·109 higher than the desorption probability. The values obtained for the KA and KD are the same as those of the other aptamers in the PCA3-specific aptamer (Luo, 2014).
Conclusion
The feasibility learning results of the electrochemical detection of lncRNA PCA3 prostate cancer marker in buffer solution through the application of specific redox-labelled aptamer are promising. It proves that the electrochemical aptasensing simple idea based on alterations in the transfer of charge between the aptamer redox label and the electrode when the aptamer-totarget binding can be used in the detection of large biomolecules like PCA3. Both the methods of impedance spectroscopy and the cyclic voltammograms gave allowance in detecting the PCA in from 1 µg/mL down to 0.1 ng/mL concentrations. The sensitivity of detection is high, with the LOD values for EIS method at 0.03 ng/ml and for the CV technique, at 0.04-0.09 ng/ml. These results are the same as the 0.26 pm and 0.35-0.78 pm in that order, with an assumption that the PCA3 fragment size of 277nt is estimated at 87 kda. The kinetic research of binding PCA3 to our aptamer gave allowance for the examination of the affinity constant at 4.10-10 molar which indicates that there is a high specific binding reaction like the one noticed between antigen to antibody interactions. There was no response in the negative control experiments.
References
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