Identification of acetaldehyde based on plasmonic patterns of a gold nanostructure conjugated with chromophore and H2O2: a new platform for the rapid and low-cost analysis of carcinogenic agents by colorimetric affordable test strip (CATS).

Acetaldehyde, a prevalent carbonyl compound in fermented foods, poses challenges in various applications due to its reactivity. This study addresses the need for efficient acetaldehyde detection methods across biotechnological, environmental, pharmaceutical, and food sectors. Herein, we present a novel colorimetric/UV spectrophotometric approach utilizing gold nanoparticles (AuNPs), particularly gold nano-flowers (AuNFs), for sensitive acetaldehyde identification. The method exhibits a notable sensitivity, detecting acetaldehyde at concentrations as low as 0.1 µM. The mechanism involves the interaction of acetaldehyde molecules with AuNFs, leading to a significant change in the absorbance spectrum, which serves as the basis for detection. Moreover, its applicability extends to human biofluids, notably urine samples. Integration with a cost-effective one-drop microfluidic colorimetric device (OD-µPCD) enables the development of an affordable test strip (CATS). This semi-analytical device, employing a multichannel OD-µPCD, facilitates real-time analysis of acetaldehyde in human samples. Our findings demonstrate the pioneering utilization of AuNPs for selective and sensitive acetaldehyde detection, promising advancements in environmental and occupational safety standards, and laying a foundation for enhanced detection and monitoring of related volatile organic compounds (VOCs).

Optimization of a silver-nanoprism conjugated with 3,3',5,5'-tetramethylbenzidine towards easy-to-make colorimetric analysis of acetaldehyde: a new platform towards rapid analysis of carcinogenic agents and environmental technology.

Acetaldehyde acts as an important mediator in the metabolism of plants and animals; however, its abnormal level can cause problems in biological processes. Although acetaldehyde is found naturally in many organisms, exposure to high concentrations can have effects on the eyes, respiratory system, etc. Due to the importance of detecting acetaldehyde in environmental samples and biofluids, determination of its concentration is highly demanded. There are some reports showing exposure to high concentrations of acetaldehyde for a long time can increase the risk of cancer by reacting with DNA. In this work, we presented a novel colorimetric method for rapid and sensitive detection of acetaldehyde with high reproducibility using different AgNPs with various morphologies. The redox reaction between AgNPs, 3,3',5,5'-tetramethylbenzidine (TMB) solution, and analytes endows a color change in 15 minutes that is detectable by the naked eye. UV spectrophotometry was further used for quantitative analysis. An iron mold with a hexagonal pattern and liquid paraffin were also used to prepare the paper-based microfluidic substrate, as a low cost, accessible, and rapid detection tool. Different types of AgNPs showed different lower limits of quantification (LLOQ). The AgNPs-Cit and AgNPrs could identify acetaldehyde with linear range of 10-7 to 10 M and an LLOQ of 10-7 M. The AgNWs showed the best color change activity with a linear range 10-5 to 10 M and the lowest diagnostic limit is 10-5 M. Finally, analysis of human biofluids as real samples were successfully performed using this system.

Colorimetric and naked-eye detection of arsenic(iii) using a paper-based microfluidic device decorated with silver nanoparticles.

Arsenic (As) as a metal ion has long-term toxicity and its presence in water poses a serious threat to the environment and human health. So, rapid and accurate recognition of traces of As is of particular importance in environmental and natural resources. In this study, a fast and sensitive colorimetric method was developed using silver nano prisms (Ag NPrs), cysteine-capped Ag NPrs, and methionine-capped Ag NPrs for accurate detection of arsenic-based on transforming the morphology of silver nanoparticles (AgNPs). The generated Ag atoms from the redox reaction of silver nitrate and As(iii) were deposited on the surface of Ag NPrs and their morphology changed to a circle. The morphological changes resulted in a change in the color of the nanoparticles from blue to purple, which was detectable by the naked eye. The rate of change was proportional to the concentration of arsenic. The changes were also confirmed using UV-Vis absorption spectra and showed a linear relationship between the change in adsorption peak and the concentration of arsenic in the range of 0.0005 to 1 ppm with a lower limit of quantification (LLOQ) of 0.0005 ppm. The proposed probes were successfully used to determine the amount of As(iii) in human urine samples. In addition, modified microfluidic substrates were fabricated with Ag NPrs, Cys-capped Ag NPrs, and methionine-capped Ag NPrs nanoparticles that are capable of arsenic detection in the long-time and can be used in the development of on-site As(iii) detection kits. In addition, silver nanowires (AgNWs) were used as a probe to detect arsenic, but good results were not obtained in human urine specimens and paper microfluidic platforms. In this study, for the first time, AgNPs were developed for optical colorimetric detection of arsenic using paper-based microfluidics. Ag NPrs performed best in both optical and colorimetric techniques. Therefore, they can be a promising option for the development of sensitive, inexpensive, and portable tools in the environmental and biomedical diagnosis of As(iii).

Platinum(II) and Copper(II) complexes of asymmetric halogen-substituted [NN'O] ligands: Synthesis, characterization, structural investigations and antiproliferative activity.

In order to better understand the effect of structure, halogen substitution, metal ions and ligand flexibility on antiproliferative activity, eight Cu(II) complexes and eight Pt(II) complexes were obtained of 2,4-X1,X2-6-((pyridine-2-ylmethylamino)methyl)phenol and 2,4-X1,X2-6-((pyridine-2-ylmethylamino)ethyl)phenol (where X is Cl, Br, or I) ligands. The compounds were characterized with various techniques, such as FT-IR, NMR, elemental analysis and single-crystal X-ray diffraction (SCXRD). The X-ray structures showed that ligand acts as a bidentate and tridentate donor in Cu(II) and Pt(II) complexes, respectively. This difference in structures is due to the use or non-use of base in the preparation of complexes. Also, complexation of Cl2-H2L1 with CuCl2·2H2O gives two different types of structures: polymer (Cl2-H2L1-Cupolymer) and dimer (Cl2-H2L1-Cudimer), according to the crystal color. In addition, 1H NMR spectrum for platinum complexes display two set of signals that can be attributed to the presence of two isomers in solution. All complexes induced moderate to high reduction in A2780 and HCT116 cancer cell viability. However, only complexes bearing iodo- substituted in ligands exhibited significantly low cytotoxicity in normal fibroblasts when compared with cancer cell lines. The antiproliferative effect exhibited by I2-H2L2-Cu complex in A2780 cell line was due to induction of cell death mechanisms, namely by apoptosis and autophagy. I2-H2L2-Cu complex does not cause DNA cleavage but a slight delay in cell cycle was observed for the first 24 h of exposition. High cytotoxicity was related with the induction of intracellular ROS. This increase in intracellular ROS was not accompanied by destabilization of the mitochondrial membrane which is an indication that ROS are being triggered externally by I2-H2L2-Cu complex and in agreement with an extrinsic apoptosis activation. I2-H2L2-Cu complex has a pro-angiogenic effect, increasing the vascularization of the CAM in chicken embryos. This is also a very important characteristic in cancer treatment since the increased vascularization in tumors might facilitate the delivery of therapeutic drugs. Taken together, these results support the potential therapeutic of the I2-H2L2-Cu complex.

Reliable recognition of DNA methylation using bioanalysis of hybridization on the surface of Ag/GQD nanocomposite stabilized on poly (ß-cyclodextrin): A new platform for DNA damage studies using genosensor technology.

Due to the role of DNA methylation in causing cancer in the present study, an innovative and inexpensive method was designed for the sensitive detection of DNA methylation. The silver-graphene quantum dots (Ag/GQDs) nano ink with high electrical conductivity was used as a substrate for genosensor fabrication toward identification of DNA hybridization. Also, poly (ß-cyclodextrin) (p[ß-CD]) has been used as a biointerface for the stabilization of Ag/GQD nano ink. The thiolated pDNA strand (5'-SH-TCCGCTTCCCGACCCGCACTCCGC-3') (as bioreceptor element) was fixed on the substrate and hybridized with methylated (5'-GC(M)GGAGTGC(M)GGGTC(M)GGGAAGC(M)GGA-3') and unmethylated (5'-GCGGAGTGCGGGTCGGGAAGCGGA-3') cDNAs, as target sequences were studied using electroanalysis methods. Under optimal conditions and using electrochemical techniques, the linear range was 1 am to 1 pm with LLOQ of 1aM. Finally, the designed DNA genosensor was used for detection of DNA methylation in human plasma samples and can be used to detect methylation in patient samples. It is expected that the designed DNA-based biodevice will be used to early stage diagnosis of cancer using monitoring of DNA methylation. Also, this type of genosensor can be used for epigenetic studies in the near future.

Electrochemical immunoplatform to assist in the diagnosis of oral cancer through the determination of CYFRA 21.1 biomarker in human saliva samples: Preparation of a novel portable biosensor toward non-invasive diagnosis of oral cancer.

In this study, a novel, low-cost, and flexible paper-based electrochemical immunosensor was developed for the bioanalysis of Cyfra 21.1 biomarker in human saliva samples by using stabilization of synthesis Ag nano-ink on the surface of paper using pen-on-paper technology. The employed electrochemical techniques for the evaluation of immunoplatform performance were differential pulse voltammetry and chronoamperometry. Also, the prepared immunosensor showed great ability in the determination of Cyfra21.1 in human saliva specimens. Under the optimized conditions, the obtained linear range was from 0.0025 to 10 ng/mL, and the obtained LLOQ was 0.0025 ng/mL. The developed immunosensor is easy to prepare, sensitive, cost-effective, portable, and simple. So proposed immunoplatform can be an accomplished biodevice in clinical laboratories. The proposed paper-based immunosensor could be a hopefully new and cheap tool for the diagnosis of other biomarkers. Also, the prepared immunosensor showed great ability in the determination of Cyfra21.1 biomarker in human saliva specimens.

A microfluidic paper-based colorimetric device for the visual detection of uric acid in human urine samples.

The monitoring of uric acid (UA) as a clinically relevant toxic biomolecule is of particular importance for the diagnosis of various syndromes and for the monitoring of patients undergoing chemotherapy or radiation therapy. Owing to its speed, low consumption of materials, high sensitivity, convenience, and the easy detection of color changes, colorimetric methods have attracted a lot of attention compared to other methods. The use of nanoparticles has been suggested for the non-enzymatic POC detection of biological molecules such as UA. Here, a sensitive, quantitative, and rapid diagnostic method for UA using silver nanoparticles (AgNPs) is reported. The main purpose of this work is to introduce a suitable tool for future studies based on various types of AgNPs for the on-site detection of clinical samples and biomarkers using portable devices. In the present study, a novel µPCD made to measure UA was used in human urine samples. AgNPs with their peroxidase-like activity led to the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and a bluish-green color upon the decomposition of hydrogen peroxide to ˙OH. UA also reduced the oxidized TMB. The proposed method showed linear responses from 500 to 10 000 µM (using silver citrate nanoparticles (Ag-Cit)), 50 to 10 000 µM (using Ag NPrs and Au@AgNPs), and 1 to 10 000 µM (using Ag NWs). The lower limits of quantification of the proposed method for the detection of UA using Ag-Cit, Ag nanoprisms, Au@Ag core-shell nanoparticles, and Ag nanowires were 500, 50, 50, and 1 µM, respectively. As a result, the proposed assay system could potentially be utilized to detect UA in human urine samples.

Cytotoxic oxidovanadium(IV) complexes of tridentate halogen-substituted Schiff bases: First dinuclear V(IV) complexes with O → VIV = O → VIV = O core.

The reaction of potentially N,N,O-tridentate Schiff base ligands, Cl-LH, Br-LH, BrCl-LH and H-LH, with [VIVO(acac)2] in 2:1 ratio in methanol gave the corresponding mononuclear and dinuclear oxidovanadium(IV) complexes, VO(Cl-L)2 (1), VO(Br-L)2 (2), [(BrCl-L)2(H2O)V(µ-O)VO(BrCl-L)2] (3) and [(H-L)2(H2O)V(µ -O)VO(H-L)2] (4), in good yields. The ligands and complexes were fully characterized by elemental analysis and FT-IR spectroscopy. The ligands were also characterized by 1H NMR spectroscopy. The oxidation state of V(IV)O with d1 configuration in all synthesized complexes was confirmed by EPR. Moreover, the structures of 2 and 3 were determined by X-ray diffraction (XRD) analysis which revealed them as mono- and dinuclear vanadium(IV) complexes, respectively, with the ligands coordinated as bidentate chelates. The structure of 3 represents the first example of dinuclear V(IV) complex with O â†’ VIV = O â†’ VIV = O core (Cambridge Structural Database (CSD)​, version 5.42, update of May 2021). The cytotoxicity of ligands and complexes was evaluated towards ovarian (A2780), breast (MCF7) and prostate (PC3) cancer cells at 48 h. While ligands showed modest IC50 values (>42 µM), all complexes turned out to be effective in the range 3.9-17.2 µM. In particular, A2780 and MCF7 cell lines were the most sensitive to the newly synthesized V(IV)O complexes.

Chemical binding of pyrrolidinyl peptide nucleic acid (acpcPNA-T9) probe with AuNPs toward label-free monitoring of miRNA-21: A novel biosensing platform for biomedical analysis and POC diagnostics.

miRNAs are attractive factors in cancer research studies due to their important roles for regulating of gene expression. Because of miRNA-21 expression surplus in many types of cancers, so accurate identification is important. Increasing efforts have caused different methods to improve the sensitivity and specificity of detection. Present study is an attempt to report a new electrochemical label-free PNA-based bioassay for detection of miRNA-21. In this study, gold electrode was modified by gold nanoparticles to improve a functional PNA-based biosensor. The EDS and field emission scanning electron microscope (FE-SEM) were used to detect fabrication of biosensor. The electrochemical behavior of sensor was evaluated after inserting of acpcPNA probes and miRNA-21 on the stucture of electrode and analyzed essential parameters such as various concentration of target miRNA, hybridization time, reproducibility, stability, and applicability. The results of study demonstrated that engineered biosensor was successfully fabricated. The findings showed the highest amount of current in 5 minutes hybridization time, with suitable reproducibility and stability. This innovative miRNA-based biosensor presents a sensitive and specific method in fast and may be lab-on chip assay in future.

Flexible paper-based label-free electrochemical biosensor for the monitoring of miRNA-21 using core-shell Ag@Au/GQD nano-ink: a new platform for the accurate and rapid analysis by low cost lab-on-paper technology.

miRNA-21 is one of the most famous and prominent microRNAs that is important in the development and emergence of cancers. So, the sensitive and selective monitoring of miRNA-21 as a very common biomarker in cancer treatment is necessary. In this work, a novel paper-based electrochemical peptide nucleic acid (PNA) sensor was developed for the detection of miRNA-21 in human plasma samples by using Ag@Au core-shell nanoparticles electrodeposited on graphene quantum dots (GQD) conductive nano-ink (Ag@Au core-shell/GQD nano-ink), which was designed directly by writing pen-on paper technology on the surface of photographic paper. This nano-ink has a great surface area for biomarker immobilization. The prepared paper-based biosensor is very small and cheap, and also has high stability and sensitivity. Hybridization of PNA was measured using various electrochemical techniques, such as cyclic voltammetry (CV), square wave voltammetry (SWV) and chronoamperometry (ChA). FE-SEM (Field Scanning Electron Microscope), TEM (Transmission Electron Microscope), EDS and DLS (Dynamic Light Scattering) tests were performed to identify the engineering safety sensor. Under optimal conditions, the linear range for the calibration curve was from 5 pM to 5 µM, and the achieved LLOQ was 5 pM. The obtained results recommended that the proposed bioassay might be suitable for an early diagnosis of cancer based on the inhibition of the expression of miRNA-21, which activates the enzyme caspase and accelerates apoptotic proteins and death in tumor cells.

Architecture of a multi-channel and easy-to-make microfluidic paper-based colorimetric device (µPCD) towards selective and sensitive recognition of uric acid by AuNPs: an innovative portable tool for the rapid and low-cost identification of clinically relevant biomolecules.

Uric acid (UA) is the end product of purine metabolism. Uric acid is usually excreted in the urine, but its abnormal increase and toxic amount can lead to diseases such as gout, hyperuricemia, Lesch-Nyhan syndrome, and cardiovascular disease. On the other hand, UA reduction can lead to neurodegenerative diseases such as sarcoma, glioblastoma, Hodgkin, and etc. Therefore, rapid identification of UA is of great importance. In this work, a simple, portable, inexpensive, and fast microfluidic paper-based colorimetric sensor based on the color change in the presence of UA by using AuNPs was developed. The results can be easily identified with naked eye and further confirmed by UV-vis spectrophotometry. In this method, iron pattern and fiberglass paper were used to construct diagnostic areas and hydrophilic microfluidic channels. We greatly reduced the preparation time of this pattern using a magnet (about three minutes). In this work, four types of nanoparticles with different lower limit of quantification (LLOQ) were used. Linear range of 10-6 to 10-3 M and LLOQ of 10-6 M were obtained for the determination of uric acid using AuNPs-CysA as optical probe. Also, by AuNPs as optical probe a linear range of 10-4 to 10-2 M and the obtained LLOQ was 10-4 M. Finally, by AuNFs as optical probe linear range from 10-6 to 10-2 M and 5 × 10-5 to 10-2 M along with LLOQ of 10-6 and 5 × 10-5 M, respectively. The designed system successfully studied in human urine samples.

Optimized DNA-based biosensor for monitoring Leishmania infantum in human plasma samples using biomacromolecular interaction: a novel platform for infectious disease diagnosis.

Leishmania parasite identification is very important in clinical studies of leishmaniasis and its diagnosis. Though there are various clinical and epidemiological approaches to identifying Leishmania infantum, due to some limitations of the traditional methods, sensitive and specific techniques are needed and are in great demand. To achieve selective and rapid detection, a sensitive signal transducer with high surface area is necessary. In this work, a new paper sensor was fabricated using silver nanoprisms electrodeposited on the GQD conductive nano-ink (Ag NPr/GQDs nano-ink). A high surface area and suitable interface for anchoring biomolecules was achieved by electrodepositing gold nanoparticles (AuNPs) functionalized with cysteamine (AuNPs-CysA) on the surface of the paper sensor altered by Ag NPr/GQDs nano-ink. To prepare a sensitive and selective bio-device for the recognition of Leishmania in human plasma specimens, a DNA-thiol probe was stabilized on the surface of the platform. Hybridization of DNA was evaluated by chronoamperometry (ChA). The engineered DNA-based paper biosensor showed high sensitivity and selectivity for the identification of Leishmania genomic DNA. Under optimum circumstances, a linear range was obtained using photographic paper from 1 µM to 1 zM and an ivory sheet from 1 nM to 1 zM. The lower limits of quantitation (LLOQ) on the photographic paper and ivory sheet were 1 zM. In addition, the designed DNA-based biosensor revealed well-defined performance in the recognition of mismatched sequences (single base, two base and three base mismatches) and selectivity.

A novel immunosensor for the monitoring of PSA using binding of biotinylated antibody to the prostate specific antigen based on nano-ink modified flexible paper substrate: efficient method for diagnosis of cancer using biosensing technology.

Prostate cancer is the most significant reason for deaths in men, outside of lung cancer. The clinical examination of cancer proteins or biomarkers is extremely significant in early examination and monitoring of recurrence of disease after treatment. Biomarkers have expanded great clinical significance owing to their extensive spectra in the identification, elimination, early diagnosis and cure of cancer. In this work, novel, ultrasensitive sandwich-type portable bio device based on citrate-capped silver nanoparticles (Citrate-AgNPs) modified graphene quantum dots (GQDs) nano ink for detection of Prostate specific antigen (PSA) was fabricated. Functionalized cysteamine with gold nanoparticles (Cys-AuNPs) was also utilized to amplify the signal. It provides a good and high external area for the immobilization biotinylated antibody of PSA in the large amount. For the first time, citrate-AgNPs-GQDs nano ink was directly written on the cellulose paper surface (ivory sheet and photographic paper) and modified by Cys-AuNPs. So, final structure of the immunodevices was completed after including of Ab1 and PSA (antigen). The immunosensors were used for the recognition of PSA by using DPVs (differential pulse voltammetry) technique. The obtained low limit of quantification (LLOQ) of the first immunodevice (ivory sheet/Citrate AgNPs-GQDs nano-ink/CysA-Au NPs/Ab1/BSA/PSA/Ab2) was 0.07 µg/L and the linear range for the calibration plot was from 0.07 to 60 µg/L. Also, the achieved LLOQ of the second immunodevice (photographic paper/Citrate AgNPs-GQDs nano-ink/Cys-Au NPs/Ab1/BSA/PSA/Ab2) was 0.05 µg/L with the linear range of 10 to 0.05 µg/L. It is noteworthy that, proposed immunoassay was effectively utilized to the monitoring of PSA glycoprotein in unprocessed human plasma sample. Obtained results show that the constructed immunosensor is able to apply as portable bio device for the clinical analysis of PSA in human plasma samples.

Binding of pDNA with cDNA using hybridization strategy towards monitoring of Haemophilus influenza genome in human plasma samples.

Haemophilus Influenza leads to respiratory infections such as sinusitis, acute otitis media, pneumonia and bronchitis. In addition, it causes invasive infections such as cellulite, septic arthritis, and meningitis. Therefore, quick and sensitive detection of H. influenza is of great importance in medical microbiology. In this study, a novel DNA-based bioassay was developed to the monitoring of Haemophilus influenza genome in human plasma samples using binding of pDNA with cDNA. DNA hybridization strategy was used to investigation of DNAs binding. For this purpose, silver nanoparticle doped graphene quantum dots inks functionalized by D-penicillamine (Ag NPs-DPA-GQDs) were synthesized and deposited on the surface of glass carbon electrode (GCE). Also, gold nanoparticles functionalized with cysteamine (CysA-AuNPs) were deposited on the surface of the Ag-DPA-GQDs modified GCE. Afterward, thiolated DNA probe was immobilized on the surface of the modified electrode. DNA hybridization was monitored using square wave voltammetry (SWV) technique. Engineered genosensor indicated good performance with high specificity and sensitivity for detection of Haemophilus influenza genome. Under optimal conditions, linear range and low limit of quantitation (LLOQ) were obtained as target concentrations ranging from 1 pM-1 ZM and 1 ZM, respectively. The designed biosensor also showed high capability of discriminating one-base, two-base and three-base mismatched sequences. Also, the prepared genosensor could be easily regenerated and reused to evaluate hybridization process.

Paper based immunosensing of ovarian cancer tumor protein CA 125 using novel nano-ink: A new platform for efficient diagnosis of cancer and biomedical analysis using microfluidic paper-based analytical devices (µPAD).

Ovarian cancer is the first and most important cause of malignancy death in women. Mucin 16 or MUC16 protein also known as carcinoma antigen 125 (CA 125) is the most commonly used glycoprotein for early stage diagnosis of ovarian cancer. In this work, a novel paper-based bio-device through hand writing of Ag/RGO (silver nanoparticles/reduced graphene oxide) nano-ink on the flexible paper substrate using pen-on-paper technology was developed. The prepared interface was used to the recognition of CA 125 protein in human biofluid. For this purpose, Ag/rGO nano-ink was synthesized by deposition of Ag nanoparticles onto graphene oxide sheets and the reduction of graphene oxide to rGO simultaneously. Conductivity and resistance of conductive lines were studied after drawing on photographic paper. Subsequently, to prepare a new and unique immuno-device, paper electrode modified by cysteamine caped gold nanoparticles (CysA/Au NPs) using electrochemical techniques. CysA is bonded by sulfur atoms with Au (CysA/Au NPs), and from the amine group with hydroxyl and carboxyl groups of Ag/RGO nano-ink deposited on the surface of paper-based electrodes (CysA/Au NPs/Ag-rGO). Then, anti-CA 125 antibody was immobilized on the electrode surface through Au NPs and CA 125 positively charged amine groups interaction. Atomic force microscopy, Transmission electron microscopy, Field emission scanning electron microscopy, and dynamic light scattering, were performed to identify the engineered immunosensor. Using chronoamperometry technique and under the optimized conditions, the low limit of quantitation (LLOQ) for the proposed immunoassay was recorded as 0.78 U/ml, which this evaluation was performed at highly linear range of 0.78-400 U/ml. The high sensitivity of the electrochemical immunosensor device is indicative of the ability of this immuno-device to detect early stages ovarian cancer.

Immunosensing of breast cancer tumor protein CA 15-3 (carbohydrate antigen 15.3) using a novel nano-bioink: A new platform for screening of proteins in human biofluids by pen-on-paper technology.

Early stage diagnosis of breast cancer by monitoring the proteins in human biological fluids is the best method and the first section toward efficient therapy, delaying metastasis and hindrance mortality. In this study, graphite ink was synthesized and combined with CA 15-3 antibody in order to develop a novel immunosensor for detection of CA 15-3, breast cancer biomarker. Conductivity of bioink was examined by designing various conductive patterns on paper using a rollerball pen. Electrochemical behavior of engineered immunosensor was evaluated through employing sensitive diagnostic technique, DPV (differential pulse voltammetry). Under optimal conditions, the linear concentration range and low limit of quantification (LLOQ) of designed immunosensor was 15-250 U/mL and 15 U/mL, respectively. The process was successfully applied to assay of breast cancer biomarker (CA15-3) in unprocessed human plasma specimens.