Early stage evaluation of colon cancer using tungsten disulfide quantum dots and bacteriophage nano-biocomposite as an efficient electrochemical platform

Recently, biosensors have become popular analytical tools for small analytes due to their high sensitivity and wide analytical range. In the present work, development of a novel biosensing method based on tungsten disulfide quantum dots (WS2 QDs)-Au for rapidly and selectively detecting c-Met protein is introduced. As a proof of concept, M13 bacteriophage-based biosensors were used for the electrochemical detection of c-Met protein as a colon cancer biomarker. The M13 bacteriophage (virus), as the biorecognition element, was immobilized on glassy carbon electrodes which were modified by WS2 QDs-functionalized gold nanoparticles. The stepwise presence of the WS2 QDs, gold nanoparticles, and immobilized phage on glassy carbon electrodes were confirmed by scanning electron microscope (SEM) and square wave voltammetry (SWV) technique. The designed biosensor was applied to measure the amount of c-Met protein in standard solutions, and consequently the desirable detection limit of 1 pg was obtained. Finally, as a proof of concept, the developed platform was used for the evaluation of c-Met protein in serum samples of colon cancer-suffering patients and the results were compared with the results of the common Elisa kit. As an interesting part of this study, some concentrations of the c-Met protein in colon cancer serum samples which could not be determined by Elisa, were easily analyzed by the developed bioassay system. The developed bioassay system has great potential to application in biomedical laboratories.


Introduction
According to reports of the WHO, approximately 9 million people died in 2015 from a variety of cancers (Pourakbari et al. 2020). In recent decades, the rates of death from cancer malignancies have sharply increased. However, by improving the early stage detection processes, the cancer mortality rates could be reduced significantly (Fitzgerald 2020). The most routinely employed methods for colon cancer diagnosis, including colonoscopy, fecal immunochemical test (FIT) (Gupta et al. 2013), and sigmoidoscopy (Winawer et al. 1993), are based on tissue sampling, imaging, etc. (Rathore et al. 2013). These approaches have several disadvantages, such as invasive nature, time-consuming processes, high cost, and especially insufficient sensitivity for early detection of malignancies (Grossman et al. 1989). On the other hand, the application of biosensors (as new detection devices), and their combination with advanced nanomaterials (Jaymand 2011;Jafarizad et al. 2018) and biomaterials (Massoumi et al. 2020) for early stage and precise diagnosis of cancers has increased nowadays in the biomedical research areas (Pourakbari et al. 2019;Isildak et al. 2020;Khalilzadeh et al. 2019Khalilzadeh et al. , 2017. Biosensors offer some benefits including; ultrahigh sensitivity, desirable selectivity, cost-effectiveness, real-time monitoring, and fast response time. These features make biosensors potent, on-desk, and ready-to-use devices for clinical analyses, which also have a robust capacity for both in vitro and in vivo applications for routine clinical laboratory tests (Soleymani and Li 2017;Hasanzadeh et al. 2018;Manzoori et al. 2012;. The lack of sufficient sensitivity to low concentrations of cancer biomarkers in unprocessed blood or serum in the past decades has forced the development of new strategies, such as the merging of nanotechnology and electrochemical biosensors that can partially overcome this problem. The upgrading of electrochemical nano-biosensors seems to be a problem-solving approach with regards to sensitivity, speed, and selectivity (Wang et al. 2013). In addition, electrochemical sensors and biosensors have significant role in biomedical and pharmaceutical analysis (Saghatforoush et al. 2009;Khalilzadeh et al. 2011Khalilzadeh et al. , 2015. Quantum dots are a subcategory of semiconductors, in which, they have been used as electrochemical modifiers for improving the sensitivity of sensors and specially biosensors (Bera et al. 2010;Jalili and Khataee 2019). Tungsten (W), as an electroactive metal, and its combination with sulfide compounds make an advanced material with high surface area and acceptable electrical conductivity for application as modifier in electrochemical-based biosensors (Pumera and Loo 2014). The electrical conductivity feature of WS 2 could be further improved by merging WS 2 with conductive compounds, such as metallic nanoparticles, such as gold, silver, and platinum via onestep or sequential electrodeposition processes (Polyakov et al. 2014;Khataee et al. 2018). In this study, we applied AuNPs, which were used as a bi-proposed layer. First, they were used for improving the electrical conductivity of the developed platform, and second, they were applied for bioconjugation of bioreceptors on the designed electrochemical platform.
Some receptor tyrosine kinases (RTKs) are proto-oncogenes that are involved in the activation process of downstream signaling molecules, which play a part in cell proliferation, migration, and survival (Safaie Qamsari et al. 2017). Among the most important receptors of the RTKs are c-Kit (also known as tyrosine-protein kinase KIT), Ephrin receptor, Platelet-derived growth factor (PDGF), fms-like tyrosine kinase 3 (Flt3; which in fact belongs to the receptor tyrosine kinase class III), and tyrosine-protein kinase Met (c-Met) (Ma et al. 2003). Binding of c-Met to its ligand, namely, the hepatocyte growth factor (HGF) receptor, initiates a chain of intracellular signaling pathways that interfere with the embryogenesis as well as the process of wound healing in normal cells. Nevertheless, the aberrant HGF/c-Met signaling in malignant cells, which is almost attributed to the mutations, overexpression, and amplification of the c-Met gene, results in the progression of the tumor through the stimulation of PI3K/AKT, MAPK/ERK, JAK/STAT, Wnt/β-catenin, as well as some other signaling pathways (Birchmeier et al. 2003). Fundamental roles of the c-Met (mesenchymal epithelial transition factor) have been investigated in numerous researches involving the spreading and metastasis of many various malignancies in humans, such as pancreas, colon, stomach, breast, prostate, bones, liver, and ovary (Ma et al. 2003).
Recently, some unique features, such as high specificity for target molecules, temperature stability, and easy production procedures, have led to the increased use of bacteriophages in various fields. The application of bacteriophages in biotechnology and biosensors has increased during the last decades (Gervais et al. 2007). Because of the declared top features of bacteriophages, we used these bioreceptors on WS 2 -AuNPs platform for the detection of c-Met protein.
In this project, WS 2 QDs were cast-dried on the GCE, and then the AuNPs were electrochemically deposited on the modified electrode. Following, the c-Met bacteriophage, as the biorecognition element was immobilized on the modified electrode, and was finally applied for electrochemical evaluation of c-Met protein as a colon cancer biomarker. As far as we are aware, this is the first report on the application of nanocomposition of WS 2 QDs, AuNPs, and bacteriophage for early stage evaluation of colon cancer.

Apparatus and procedures
Autolab potentiostat/galvanostat was utilized to perform electrochemical measurements including electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), square wave voltammetry (SWV), and cyclic voltammetry (CV), for all of which the NOVA 1.8 software was used for data processing. Shimadzu 8101 M FTIR spectrophotometer (Shimadzu, Kyoto, Japan) was employed to perform Fourier transform infrared (FTIR) experiments. Preparation of the FTIR discs was performed by grinding appropriate sample contents with KBr salt. A VEGA TESCAN (Czech Republic) instrument was utilized to take the scanning electron microscopy (SEM) images illustrating the stepwise modifications on glassy carbon electrode (GCE). Initially, exposure of the samples to the electron bombardment with gold atoms was performed under vacuum condition, and then the samples were moved to SEM chamber for taking the images.

Preparation of WS 2 QDs and AuNPs
To this end, 1 g from tungsten disulfide powder was added to 100 mL of DMF, following which a solution was prepared and sonicated for 3 h. Later, stirring of the collected suspension was performed in a sealed container for 6 h at 140 °C. Proof of the successful production of the WS 2 QDs was the observed yellow solution over the sediments. The supernatant was vaporized under vacuum condition at a specific temperature and the debris was re-suspended in double distillated water for further applications (Xu et al. 2015). Consequently, the yellowish tungsten sulfide nanosheets were obtained and confirmed by FTIR.
The AuNPs were prepared using in situ electrodeposition process (Chiang et al. 2019). In brief, a solution of 0.005 M of HAuCl 4 and 0.1 M of KCl in deionized water was prepared. A portion of the obtained suspension was transferred onto the electrochemical cell and CV technique was applied for electrosynthesis process (the potential was swiped between − 0.5 and 0 V, scan rate 0.05 V/S, for 25 cycles).

Phage biopaning and amplification
In this study, 500 mL of phagemid library was added to cultured media containing 200 mL of 2XYT medium and ampicillin (120 mg/mL), by the time the optical density of the suspension at 600 nm (i.e., OD 600) achieved ≈ 0.4-0.5. Then, approximately 5 × 10 11 plaque-forming units (PFU) from the helper phage M13K07 were supplemented to each milliliter of the bacterial culture, where they were incubated for 30 min at 37 °C, and later for another 30 min at 37 °C on a shaker (at 100 rpm).
Consequently, the bacteria were centrifuged (10 min, 4 °C, at 4000 rpm) and dispersed in 100 mL of fresh 2XYT, which contained both ampicillin (120 mg/mL) and kanamycin (50 mg/mL). Next, incubation at 30 °C was performed for 16 h with shaking (200 rpm). Afterwards, purification of the phage was performed in line with the PEG/NaCl precipitation procedure ).

Electrode preparation steps and sequential modifications
The GCE was polished with alumina powder followed by electrochemical cleaning, which was processed in 0.5 M H 2 SO 4 using CV technique, where the potential range was 0.0-1.5 V versus Ag/AgCl reference electrode at scan rate of 100 mVs −1 for 10 cycles and finally dried with nitrogen stream.
The electrochemical performance of each immobilization step was studied via CV technique in the electrochemical cells containing the modified working electrode, an Ag/AgCl as a reference electrode, and a platinum wire as counter electrode in At first, the WS 2 QDs were drop casted on pre-cleaned GCE. Then, AuNPs were electrochemically deposited on the WS 2 QDs via CV technique. Next, the modified electrode was incubated with M13 phage solution as the biorecognition element. Finally, the unbound phages were removed by immersing the modified electrode in phosphate buffer solution (PBS, pH = 7) for 1 min.

FTIR
The FTIR absorption peaks are illustrated in (Additional file 1: Figure S1). Based on the obtained FTIR peaks, WS 2 QDs have two characteristic absorption peaks, where the first one approximately appeared at 659 cm −1 , which was associated to the wolfram-sulfur (W-S) bond. The second one occurred at about 866 cm −1 , which was related to the disulfide (S-S) bond. Broad peaks, which appeared at 3527 cm −1 (OH bond stretching), 2933 cm −1 (methylene asymmetric stretching), 1674 cm −1 (C = O vibration), 1394 cm −1 (C-NH-C or C = N-C stretching vibration), 1257 cm −1 (C-N stretching) and 1097 cm −1 (C-NH-C or C-N stretching), showed that the WS 2 QDs were correctly synthesized. In addition, the good water solubility of the WS 2 QDs was regarded to the presence of carboxyl and hydroxyl groups (Xu et al. 2019).

Fluorescence
The photoluminescence (PL) activity of the synthesized WS 2 QDs was characterized in 100-fold diluted samples at different excitation wavelengths (300 and 350 nm). By increasing the excitation wavelengths, the PL activity increased. The fluorescence spectra are shown in (Additional file 1: Figure S2). The obtained spectra confirmed that the prepared WS 2 QDS were synthesized properly. The same spectra were obtained by others (Haddad Irani-nezhad et al. 2019).

SEM
The topography of the modified electrode surface, nanoparticles' morphology, and composition of the deposited nanomaterials were successfully evaluated by SEM. In this regard, the stepwise modifications on the working electrode were prepared and their SEM images were taken. The SEM images are illustrated in Fig. 1A-C. As demonstrated in the different panels, the size of the WS 2 QDs are approximately in the range of 25 nm and the average size of the electrodeposited AuNPs are in the range of 25-45 nm. After casting bacteriophage as biorecognition element on the modified electrode, the surface morphology was changed. Because of the small size of the phages, their unique structures weren't specified by SEM. However, through the changes in the surface characteristics, it could be concluded that the bacteriophages were desirably conjugated on the modified electrode via N-Au and S-Au bonds. Furthermore, the SEM images were validated via the Energy-Dispersive X-Ray Analysis (EDX).

EDX
The presence of tungsten (W), sulfur (S), gold (Au) and carbon (C) with W:S ratio of 1:2 was revealed by EDX patterns of WS2 QDs, as shown in Fig. 1D, E. The presence of 'C' is due to carbonic based working electrode (GCE), while the presence of gold is mostly because of gold sputtering process during sample preparation for EDX and SEM. On top of that, the correct positioning of nanoparticles (WS2 QDs and AuNPs) and biomaterials (bacteriophage) involved in the step by step modifications was approved by EDX. The EDX results are depicted in Fig. 1F. According to the results of dot mapping, W and S are next to each other, meaning that WS 2 is properly arranged. Likewise, Au is next to S, which means that the gold nanoparticles are immobilized on S. Besides, from the combination of N and Au, it can be concluded that the phages are mostly connected to gold through the amino groups.

Characterization of electrode preparation steps
All of the electrode preparation steps were approved by square wave voltammetry (SWV) technique and are presented in Fig. 2. In this regard, at first, the bare GCE was put in the electrochemical cell containing potassium ferrocyanide (2 M) and potassium chloride (5 M) solution. Then, WS 2 QDs were immobilized on the electrode by a dropcasting method, following which the modified electrode surface was again modified via gold nanoparticles through electrosynthesis. Next, the phage solution was poured onto the modified electrode with optimized concentration.

Optimization of WS 2 QDs thickness on GCE
The first step of optimization is to optimize the concentration or thickness of nanomaterials on the working electrode surface. In this regard, different volumes (1, 2, 3, 4, 5 and 10 µL, as a function of concentration) of the initial prepared suspension of WS 2 QDs were drop casted on the pre-cleaned GCE. Then, the WS 2 QDs modified electrodes were incubated at room temperature for 1 h. Following, the electrocatalytic activity of the WS 2 QDs modified electrodes were evaluated in K 4 [Fe (CN) 6 ] as standard electrochemical solution via CV technique in the potential range of − 0.5 to 1.0 V as well as scan rate of 100 mV (5 cycles were recorded). The results of the voltammograms relevant to this study are shown in Fig. 3A. The average of the recorded peak heights corresponding to each volume are calculated and the histogram of average peak heights versus applied different volumes of WS 2 QDs are presented in Fig. 3B. According to the obtained voltammograms and corresponding histogram, the volume of 3 µL was selected as the optimal volume of WS 2 QDs for further studies. Previous studies have shown that after optimizing the nanomaterial concentration or thickness on the electrode surface, any increasing of the nanomaterials' concentration or resulted thick layers could prevent redox reaction on the modified electrode or act as a semiconductor or insulating agent (Tonelli et al. 2019). Then, the incubation temperature of the WS 2 QDs on the electrode was optimized for better binding of the WS 2 QDs. For this purpose, the modified electrodes were studied at different temperatures of 37 °C, Room Temperature (RT), and 4 °C for 1 h as constant incubation time. To investigate this step, the CV technique was applied, such as the previous optimization. The obtained voltammograms are shown in (Additional file 1: Figure S3A). In addition, the average of 5 replicates of peak heights are calculated and the related histogram is depicted in (Additional file 1: Figure S3B). Based on the obtained voltammogram and linked histogram, the temperature of 37 °C was selected as the optimum temperature for further studies.
Finally, the incubation time of the WS 2 QDs on the working electrode was optimized. For this purpose, the prepared electrodes were incubated at 37 °C for 30, 60, 90, 120, and 140 min as different incubation timepoints. Again, the CV technique, such as previous optimizations, was applied for the evaluation of different incubation times. The recorded voltammograms and related histogram relevant to this study are shown in (Additional file 1: Figure S4). According to the obtained voltammograms and corresponding histogram, 60 min was determined as the optimal incubation time for further studies.
It was observed after 60 min of incubation that by increasing the incubation time, the redox peak currents did not change significantly.

Optimization of AuNPs on the WS 2 QDs modified GCE
To increase the conductivity, stabilize the WS 2 QDs, and achieve the most obtainable and stable binding of phages (as bioreceptor) on the surface of the modified electrode, we used gold nanoparticles. For this purpose, a solution of KCl at a concentration of 0.1 M and HAuCl 4 at a concentration of 0.005 M was prepared and transferred to the electrochemical cell. The modified electrode (WS 2 QDs-GCE) was inserted into the electrochemical cell, which contained the gold solution. To optimize the thickness and morphology of the gold nanoparticles on the modified electrode, CV technique was performed in various cycle numbers (10, 15, 20, 25 and 30) in the range of − 0.5 to 0.0 V, where the scanning rate was set in 0.05 V/S. Fig. 3 A Cyclic voltammograms B The histograms of WS 2 QDs concentration at different amounts (1, 2, 3, 4 and 10 µL) from the same source sample Pourakbari et al. Cancer Nanotechnology (2022) 13:7 To investigate the electrocatalytic properties of the modified electrodes (AuNPs-WS 2 QDs-GCE), the CV technique was used in the range of 0-0.3 V. The recorded voltammograms relevant to this study are shown in (Additional file 1: Figure S5A). The average of peak heights of the five replicates are calculated and the associated histogram is shown in (Additional file 1: Figure S5B). According to the obtained voltammograms and linked histogram, 25 cycles was chosen as an optimal cycle number for the electropreparation of Au layer for additional studies.

Optimization of Phage immobilization conditions
After culturing and propagating the phages, 10 µL of homogenous phage solution with the concentration of 10 6 , which is stated by the previous studies (Ertürk and Lood 2018), was dropped on the final modified electrode (AuNPs-WS 2 QDs-GCE). To optimize better binding between phage and gold, we examined the incubation temperature and time of phage binding.
To optimize the incubation temperature of phage immobilized electrodes, different temperatures of 37 °C, RT, and 4 °C, were examined for 1 h. To investigate this step, CV techniques were applied (5 electrochemical cycles were performed). The voltammograms and histogram relevant to this study are presented in (Additional file 1: Figure S6). According to the obtained voltammograms and histogram, the temperature of 4 °C was selected as the optimum temperature for the subsequent steps. Based on the acquired results, the chemical bonding between the bioreceptor and gold nano-layer was more strong and stable at 4 °C.
Next, the incubation time of phage was optimized at different timepoints of 30, 60, 120 and 180 min at 4 °C. The corresponding voltammograms and histogram are illustrated in (Additional file 1: Figure S7). Based on the calculated results, 60 min was chosen as the optimal incubation time. The lowest interaction of phages occurred at 30 and 300 min. According to the results obtained after 60 min of incubation, increasing the incubation time did not affect the interactions between the bioreceptor and gold nano-layer.

Optimization of the target protein capturing conditions
The last step of optimization is optimizing the target protein capturing conditions on the final modified electrode (phage-AuNPs-WS 2 QDs-GCE). In this regard, 10 µL of 1000 pg/mL concentration of target protein (c-Met) was immobilized on the final modified electrode, and optimization of its incubation temperature and time was performed.
Temperatures of the incubation for the final modified electrodes carrying c-Met protein were monitored at 37 °C, RT, and 4 °C for 1 h, and related voltammograms and histogram are presented in (Additional file 1: Figure S8). The incubation temperature of 37 °C was selected as the optimum temperature. According to published articles, usually the bioconjugation between protein and phage occurs at a higher temperature as compared to phage and nanomaterial binding (Singh et al. 2009).
To optimize the incubation time of the target protein, different timepoints of 30, 60, 120 and 180 min at 37 °C were applied. The resulted voltammograms and histogram are presented in (Additional file 1: Figure S9). Based on the achieved results, 60 min was applied as the optimal incubation time. It is expected that over 60 min of incubation time, damages to the protein structure may occur.

Calibration curve
After optimization of all experimental factors, the optimized system was used to investigate the different concentrations of target protein (1, 250, 500, 750 and 1000 pg/mL). To evaluate this step, the SWV technique was applied in the standard electrochemical solution in the range of − 0.1 to 0.6 V (SWV voltammograms were repeated 3 times for each concentration). The results of SWV voltammograms and histogram corresponding to the calibration curve are shown in Fig. 4. According to the calculated results, the limit of detection of the c-Met protein as a colon cancer biomarker for the designed biosensor was 1 pg/mL as well as a linear range of 1-1000 pg/mL.

Stability and specificity
To evaluate the stability of the designed biosensor, after all preparation steps (c-Metphage-AuNPs-WS 2 QDs-GCE) (protein concentration, 1 ng/mL), the SWV was applied as same as the calibration curve condition and its voltammogram was recorded for 3 times. Then it was transferred to a refrigerator (4 °C) by placing the cap on the final modified electrode. Four days later, three SWV voltammograms were recorded again under the same conditions, and the results were finally compared. The results indicated A B Fig. 4 A Square wave voltammograms B The histograms of calibration curve that the average peak height of the fifth day SWV voltammograms had increased by almost 15%, as compared to the first day, proposing that by increasing the storage period some attachments related to different electrode preparation steps may be separated and detached from the GCE surface, and consequently the electrical signals increase. According to the obtained stability outcomes, the designed biosensor is sufficiently stable to be used as a commercial biosensor.
The specificity of the designed biosensor was successfully tested in the presence of two proteins (BSA, 0.05% and ROR1, 1 ng/mL concentrations). These two proteins are generally present in human serum samples. On the other hand, previous studies have shown that the BSA protein could react with gold, silver, and platinum, which reduces the sensitivity of the designed biosensors (Saptarshi et al. 2013). For this reason, both proteins (BSA and ROR1) were immobilized on the final modified electrode (c-Metphage-AuNPs-WS 2 QDs-GCE) in the presence of 1 ng/mL of the target protein and were incubated at room temperature for 1 h. Then, the SWV voltammograms were recorded as same as the calibration curve conditions. The resulted voltammograms and histograms for BSA and ROR1 are presented in Fig. 5.

Repeatability
To evaluate the repeatability of the developed biosensor (in the presence of 1 ng/mL of protein), the average peak height of the 10 replicates was calculated. Based on the recorded electrical signals, the desirable relative standard deviation (RSD) was obtained as 6.37% for repeatability measurements.

Reproducibility
To investigate the reproducibility of the designed bioassay, the two concentrations of target protein were selected as 1000 and 750 pg/mL. Two separated GCEs were identically modified for 1000 and 750 pg/mL, individually. The corresponding SWV Real sample analysis with designed biosensor for serum samples of different human colon cancer suffering patients at different stages. A SWV voltammograms; B corresponding histogram and C results of ELISA as a positive conventional method for the same samples voltammograms were recorded 3 times at the same experimental conditions. The results showed that the proposed biosensor had adequate reproducibility for clinical and commercial approaches.

Real samples analysis by the developed biosensor and commercial Elisa Kit
Lastly, the optimized bioassay was used for the quantification of c-Met protein in the serum samples of the patients with colon cancer at different stages. For this purpose, 9 patients and 1 healthy person as negative control were examined and their serum samples were collected and diluted (1:1000) with PBS. Then, 10 µL of the diluted serum samples were placed on the modified electrodes (instead of target protein in the calibration curve), and their SWV voltammograms were recorded as same as the calibration curve. The results of this real sample analysis are shown in Fig. 6A. The histogram of the average peak height of the three replicates was calculated and is shown in Fig. 6B.
Also, the c-Met protein of the same samples were evaluated by the Elisa protocol (as a positive control of this study) and their results are presented in Fig. 6C.
Based on the obtained results of the reals samples, there is no difference between the healthy individual samples and the electrical signal of the modified electrode (phage-AuNPs-WS 2 QDs-GCE) without target protein. In other words, there is no c-Met protein in the serum sample of the healthy person to react with or be captured by the developed biosensor. Approximately the electrical signal of four patients were in the range of 250-500 pg, while five patients were in the range of 750-1000 pg in the obtained calibration curve. In some samples, Elisa was unable to respond (samples 1, 2, 3, 4, and 8). This is a sign of the inability of Elisa and the sensitivity of the designed biosensor.

Conclusion
In conclusion, a novel biosensor based on nanomaterials and with facile fabrication procedure was developed using non-lytic bacteriophage M13 (as bioreceptor) for measuring the c-Met protein. A great detection limit was exhibited by the sensor (1 pg/mL). This excellent limit of detection could be contributed to the high surface area of WS 2 QDs and phage specificity, as well as to the effective chemical binding between M13 and the surface of well-dispersed gold nanoparticles. The specificity of the phage-based biosensor was successfully examined in the presence of ROR1 and BSA proteins, where the introduced biosensor exhibited good response with neglectable interferences. Therefore, this biosensor could be introduced as a prospective analytical tool to monitor the desired protein in real samples. Moreover, good stability is displayed by the biosensor over an extensive range of temperatures and time. In addition, high sensitivity and selectivity are observed in the reported phage-based biosensor. As a proof of concept, the constructed bioassay was effectively applied for the detection of target protein in the colon cancer suffering patients. Interestingly, the results of the developed biosensor are more precise and sensitive as compared to commercial kits.