Application of μPADs in Detection of Cancer Biomarkers: History
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Contributor:
Bilge Asci Erkocyigit
,
Ozge Ozufuklar
,
Aysenur Yardim
,
Emine Guler Celik
,
Suna Timur

Microfluidics is very crucial in lab-on-a-chip systems for carrying out operations in a large-scale laboratory environment on a single chip. Microfluidic systems are miniaturized devices in which the fluid behavior and control can be manipulated on a small platform, with surface forces on the platform being greater than volumetric forces depending on the test method used. Paper-based microfluidic analytical devices (μPADs) have been developed to be used in point-of-care (POC) technologies. μPADs have numerous advantages, including ease of use, low cost, capillary action liquid transfer without the need for power, the ability to store reagents in active form in the fiber network, and the capability to perform multiple tests using various measurement techniques. These benefits are critical in the advancement of paper-based microfluidics in the fields of disease diagnosis, drug application, and environment and food safety. Cancer is one of the most critical diseases for early detection all around the world. Detecting cancer-specific biomarkers provides significant data for both early diagnosis and controlling the disease progression. μPADs for cancer biomarker detection hold great promise for improving cure rates, quality of life, and minimizing treatment costs. 

  • biomarker
  • microfluidics
  • cancer
  • detection
  • paper-based microfluidics

1. Introduction

The early detection of cancer using disease-specific biomarkers is critical for both treatment and screening. Although immunoassays are extensively used as a detection method due to their high specificity and sensitivity, some drawbacks, such as signal amplification, target labeling, and multiple washing steps, as well as the requirement for experienced technicians, create the need for these analyses to be replaced by more applicable alternative methods. paper-based microfluidic analytical devices (μPADs) overcome these operational difficulties even though their basic working principle is simple and requires small volumes because they provide a continuous process due to the reagent flow through the microfluidic channels. Although the basic operation of μPADs is simple, when other reactive solutions such as liquid samples, wash buffers, etc. are added to the cellulose membrane, the run-off of primary antibodies becomes a problem due to non-specific adsorption in these designs. The specificity of the measurement is significantly affected by the flow of the primary antibody from the test strip during analysis. The designed μPAD is intended to overcome this limitation by providing surface chemistry that will promote strong binding and provide accurate measurement of trace cancer biomarkers in the sample. For example, when the optical detection method of μPADs is investigated, the surface chemistry is first provided by adding chitosan to the surface, which provides a simple modification, and thus the analysis sensitivity is improved with more stable immobilizations with a covalent bond. For instance, Alizadeh and colleagues created a μPAD-based immunosensor for the detection of CEA. The glutaraldehyde (GA) cross-linking method was used to immobilize primary antibodies functionalized with chitosan on the paper surface. Following the incubation of the sample, a Co2(OH)2CO3-CeO2 nanocomposite functionalized with secondary antibodies was added to the sensor. The nanocomposite’s peroxidase-like activity then caused TMB oxidation in the presence of hydrogen peroxide, resulting in a color change. Sulfuric acid was then added, and the color change from blue to yellow was observed. Color can be seen with the naked eye, and quantitative results were obtained by capturing images with a smartphone and analyzing them with specialized software [1]. Wang and colleagues developed a chemiluminescence immunoassay-based PAD with an integrated magnetically actuated valve and an adjustable time controller for the rapid determination of numerous tumor markers. Photolithography was used for the patterning and fabrication of the PAD. The capture antibodies (CEA, AFP, and CA-125) were immobilized by chitosan coating and glutaraldehyde cross-linking. The time controller’s timing channel included two adjustable conductive iron bands that functioned as an electric switch. The circuit was closed, the magnetic valve was activated, and the chemiluminescence immunoassay reaction was started by connecting these bands via fluid flow. In comparison to the conventional method, the reaction based on HRP-O-phenylenediamine-H2O2 required only about 3.4 min of incubation time [2]. Timed fluid control (TFC) is a fascinating method for automating immunoassay-based PADs. TFC can be accomplished in three ways: (1) by changing the geometry of the channel, (2) by adding chemicals to generate a programmable flow delay, or (3) by using a mechanical valve to control the reaction’s state. The incorporation of superior properties of nanostructures in the biosensing process contributes to the development of functional PADs and the provision of higher analytical performance, as well as the shortage of practical applications in this field. Recent applications have clearly demonstrated that these PAD systems have the potential to be integrated into point-of-cares (POCs) (such as telemedicine and smartphone applications), and they can be improved and developed as new requirements arise. Despite the fact that research in this area is limited, this section presents important and recent strategies for the use of μPADs in cancer application [3][4] In Table 1, comparison of μPAD applications in the detection of cancer biomarkers were given in terms of target biomarker, fabrication and detection technique, surface chemistry and analytical performance parameters including linear range and limit of detection (LOD).

2. Detection of Protein Biomarkers

Proteins are biomolecules found in the body that, when expressed abnormally, can be associated with a variety of diseases, including cancer. Cancer cells can produce proteins that they can release into body fluids such as blood and urine. Cancer proteins, unlike other proteins, are difficult to identify due to their low concentration. Accordingly, single protein detection or multiple protein detection using different signal-enhanced detection strategies can be used to monitor cancer detection, cancer stage, and treatment process [4].
Wang et al. created a multi-parameter paper-based electrochemical aptasensor with high sensitivity and specificity for the simultaneous detection of CEA and neuron-specific enolase (NSE) in a clinical sample. The microfluidic channels in the system were created using wax printing, while the three-electrode system was created using screen printing. Prussian blue (PB)–poly(3,4-ethylenedioxythiophene) (PEDOT)–AuNPs nanocomposites and amino functional graphene (NG)–thionine (THI)–AuNPs were produced. These nanocomposites were used not only to boost electron transfer rates, but also to replace working electrodes in the immobilization of CEA and NSE aptamers. To detect CEA and NSE in clinical samples, a fast, simple, and label-free electrochemical method was used. To validate the clinical application of the developed aptasensor, fifteen clinical serum samples provided by the Peking University Cancer Hospital were tested using a commercially available Roche electrochemiluminescence apparatus. Under optimal conditions, the multi-parameter aptasensor showed excellent linearity in the 0.01–500 ng mL-1 range for CEA (R2 = 0.989) and 0.05–500 ng mL−1 range for NSE (R2 = 0.944), respectively. The 3/S calculated the LOD to be 2.0 pg mL−1 for CEA and 10 pg mL−1 for NSE. The price of a paper device was calculated to be around $0.12. As a result, the presented device could offer a low-cost and portable diagnostic platform for cancer biomarkers [5].
In another study, a paper-based chip immunoassay was developed to analyze AFP using the sandwich method. To capture AFP, primary antibodies are immobilized on paper using chitosan. In secondary antibodies, altered starter DNAs can trigger a hybridization chain reaction to amplify fluorescence signals for AFP. A laser-induced fluorescence detector integration was used to detect the targets. Under ideal conditions, the limit of detection for AFP in the study was 1.0 pg/mL [6].
In contrast to traditional paper-based microfluidic analytical devices, developed innovative 3D μPADs can control fluid flow dynamics in 3D using only capillary-driven flow and no external pump. A sequential and automatic multi-step ELISA test can be performed through the 3D bridge structure by simultaneously adding the sample, washing, and enzyme–substrate solutions used in the study to the system in the 3D μPAD. Thioredoxin-1 (Trx-1) was studied as a target biomarker in this 3D μPAD study, which is a new breast cancer biomarker in the literature. The results demonstrated that the 3D μPAD provides a 0–200 ng/mL detection range for Trx-1. Sera from patients and healthy people were used to confirm that this chip is suitable for commercial use and that Trx-1 can be detected accurately in real serum samples. Validation studies using ELISA revealed significant differences for this new biomarker between healthy and sick people. As a result, it is clear that the 3D μPAD created in the real-world example is a commercially extensible platform [7].
Yang and colleagues produced fully functional tests that truly achieve the goal of POC testing. The microfluidic channel was created using wax printing, while the electrodes were created using screen printing. The researchers successfully synthesized NH2-G/Thi/AuNPs nanocomposites, which were then used to modify the working electrode. The working electrode had high bioactivity after modification, indicating that it could be used to detect CEA with immobilized anti-CEA. A label-free electrochemical method that avoids labeling antigens or antibodies during measurement was used to make CEA detection faster, simpler, and less expensive. The results of the experiment revealed that the limit of detection for CEA was 10 pg mL−1 (S/N = 3) and the correlation coefficient was 0.996 in the range of 50 pg mL−1 to 500 ng mL−1 [8].
The sensitive and selective detection of PSA, a commonly used marker in the diagnosis of prostate cancer, remains challenging. A label-free μPAD for accurate electrochemical detection is reported by Wie et al. In this research, wax printing was used to create the microfluidic channel, and screen printing was used to create the triple-electrode system. By creating AuNPs/reduced graphene oxide (rGO)/thionine (THI) nanocomposites, the working electrode surface was modified to provide immobilization of the probe DNA aptamer sequence. The excellent conductivity of AuNPs and rGO also contributes to electron transfer, while THI acts as an electrochemical mediator to communicate the biological recognition between the DNA aptamer and PSA. In the study, LOD was detected at 10 ng/mL and a linear range of 0.05 to 200 ng mL−1. Real samples from Shijitan Hospital were analyzed to further investigate the applicability of the developed aptasensor in clinical settings. The results of clinical serum samples obtained from Shijitan Hospital using the proposed aptasensor were compared to reference values obtained in the hospital using the electrochemiluminescence method (Roche, USA). The analytical results were generally compared with hospital reference values, with relative errors calculated to be less than 8.81%. In conclusion, the proposed aptasensor is proposed to be used to detect PSA in clinical samples [9].
Fan et al. created a wireless POCT system that includes μPADs, an electrochemical detector, and an Android smartphone for the electrochemical detection of NSE. Nanocomposites containing amino functional graphene, thionine, and AuNPs (NH2-G/Thi/AuNPs) were used to modify PADs. It demonstrated good linearity across the concentration range from 1.0 ng mL−1 to 500 ng mL−1. The detection results were displayed in real time on the smartphone via Bluetooth connection. For a real sample analysis, the wireless POCT system also demonstrated remarkably good agreement with the ELISA method. The wireless POCT system can primarily focus on the detection of other tumor biomarkers because of its low cost, high accuracy, and low detection limit. Leading healthcare institutions as well as nations with limited resources use it. This integration of wireless systems with various storage systems can also play a significant role in telemedicine [10].
In another study, Mohamed Shehata Draz and his colleagues published a paper–plastic microchip (PPMC). The CEA and AFP amounts in a single sample were determined using the impedimetric analysis technique with the suggested PPMC. In this research, the imprinted electrolytes were designed as semicircular while developing the PPMC, and target analytes were detected by immobilizing anti-CEA and anti-AFP to the detection regions. Impedance measurements of AFP and CEA on PPMC revealed a detection limit of 102 ng mL−1 for both targets when using the multiplex detection format; when using the single analyte detection format, these limits were reduced to 10 ng/mL and 1.0 ng/mL, respectively. Thus, the sensitivity of the PPMC has been improved. The flexible PPMC system, which can detect analytes and conduct impedimetric analysis, is thought to be useful for wearable technology-based cancer detection [11].
The epidermal growth factor receptor (EGFR) was detected by a label-free method with extreme sensitivity using anti-EGFR aptamers as the biorecognition component in a study reported by Wang et al. In the device, the origami concept was applied in order to decrease sampling volumes and improve usability. The nanocomposites created by attaching the aptamers to the surface with H2-GO/THI/AuNP were added to the working electrode surface and the surface structure was changed. The detection limit for CEA was determined as 10 pg mL−1 (S/N = 3), with the R2 of 0.996 in the range from 50 pg mL−1 to 500 ng mL−1. Analyzing sera revealed an excellent correlation with the gold-standard ELISA, demonstrating the analytical accuracy of the paper-based aptasensor [12].
Based on the origami structure, Shuai Sun et al. proposed an mPAD to detect vascular endothelial growth factor C (VEGF-C). VEGF-C in serum samples was determined using an electrochemical analysis method in this research. The flow channels were created using wax printing, while the electrodes were printed on filter paper using screen printing. The working electrode was then modified with nanocomposites of methylene blue, amino functional single-walled carbon nanotubes, and gold nanoparticles (NMB/NH2-SWCNTs/ AuNPs). Thus, an mPAD with linearity ranging from 0.01 to 100 ng mL−1 (R2 = 0.988) and a detection limit of 10μg mL−1 was developed by improving the analysis process. The accuracy of the suggested device was achieved by supplying real clinical samples into the proposed system. As a result, this research provides an improved platform for real-time cancer detection [13].
The silver nanoparticle-reduced graphene oxide nanocomposite (Ag/RGO) ink created by Soodabeh Hassanpour and colleagues served as the foundation for the development of an immunosensor for a paper-based system. In this procedure, a high concentration of CA15-3 antibodies was added to the system following the immobilization of cysteamine-coupled AuNPs (CysA/Au NPs), which provide signal amplification, to the surface. The CA 15-3 biomarker immunosensor’s lower limit of quantification (LLOQ) was determined to be 15U/mL, and the calibration curve was linear in the range of 15–125 U/mL [14].
A new disposable and sensitive microfluidic paper-based electrochemical immunosensor on rGO-TEPA/Au electrode materials has been developed. With a simple and disposable paper-based microfluidic channel based on SPEs/rGO-TEPA/Au, both immunochromatography and immunofiltration were achieved simultaneously. Using portable SPEs/rGO-TEPA/Au and a simple μPAD, HRP-labeled signal antibodies (Ab2) and co-fixed gold nanorods were investigated as tracers for square wave voltammetry (SWV) detection. Due to the performance of HRP-GNRs-Ab2 captured at the detection site under optimal conditions, the system has wide linear ranges (0.01 ng mL−1–100.0 ng mL−1) with a detection limit of 0.005 ng mL−1 by using AFP as the model analyte [15].
Based on the origami principle, Yucui Jiao and colleagues created a new 3D vertical flow paper-based device (3VPD) coupled with fluorescent immunoassay that can simultaneously detect several cancer biomarkers. The device is composed of three layers: (1) sample, (2) separation, and (3) test layers so that a vertical flow immunoassay was fabricated. Fluorescent isothiocyanate (FITC)-labeled antibodies are coated on the well layer in this assay, while mouse monoclonal capture antibodies are functionalized on the test layer. The device detected three cancer biomarkers, CEA, AFP, and CA199, with detection limits of 0.03 ng/mL, 0.05 ng/mL, and 0.09, respectively [16]. It created a strategy for the instantaneous detection of multiple analytes as well as a low-cost and sensitive method for comprehensive clinical diagnosis using a single test.
Bo Dai at all. developed a flux-adaptive, self-contained microfluidic platform that includes a serological analysis platform (SAP) where CLIAs for multiple teardrop-shaped biomarkers can be run concurrently. The platform is designed to allow a low flow of sample liquid to pass through the reaction areas while speeding up the reaction as it moves between the areas. In addition, a small device for chemiluminescence detection and signal analysis has been developed. Four colorectal cancer biomarkers were immobilized to the reaction sites in the design. As a result, the microdevice added to the developed platform was used to analyze four different biomarkers at the same time. It ended up taking approximately 20 min to complete the test. CEA, AFP, CA125, and CA19-9 have detection limits of 0.89 ng mL−1, 1.72 ngmL−1, 3.62 UmL−1, and 1.05 U mL−1, respectively. Results from laboratory testing using ECLIAs based on commercial testing kits and measured using a commercial immunoassay analyzer were compared to those obtained from testing on the microfluidic platform. The calculation coefficient for the linear regression being greater than 0.9990 indicates a highly linear correlation between the SAP and the commercial immunoassay analyzer. The study in question demonstrated that the self-contained and adaptable microfluidic platform can be developed for a wide range of applications in POC-based disease health monitoring [17].

3. Detection of Nucleic Acid Biomarkers (Circulating Tumor DNA (ctDNA) and microRNA (miRNA))

The use of biofluids such as blood, saliva, and urine for cancer detection has emerged as a revolutionary technique for diagnosis and prognosis. Compared to traditional solid tissue-based biopsy tests, liquid biopsy has the advantage of being non-invasive. Therefore, it is an ideal and promising technique for monitoring and tracking cancer progression and genetic abnormalities. Liquid biopsy is a simple examination of CTCs as well as circulating tumor-derived material such as ctDNA, cfmiRNAs, and extracellular vehicles. Important information is obtained through evaluation using invasive procedures [18].
CTCs are cancer cells that have separated from the primary tumor and entered the blood system. Because CTCs are rare and highly inhomogeneous, only a small amount of sample is produced during the enrichment stage of CTC analysis. Several technologies based on CTC biophysical and biological characteristics have been developed to differentiate these cells from the background and enrich them for successive molecular or image processing analysis [18].
Exosomes are subunits of extracellular vesicles (EVs) that are essential for intercellular communication and transport of molecules from donor to recipient cells. According to recent research, exosomes have a high potential for use as novel biomarkers in liquid biopsy due to their abundance in body fluids and involvement in a variety of physiological and pathological processes [19][20].
Cell-free DNA (cfDNA), which is primarily released by hematopoietic cells, has been found for both physiological and pathological states and is currently widely used in prenatal diagnosis [18].
Small RNAs, also referred to as small noncoding regulatory RNAs, play an important function in regulating gene expression. Small RNAs are classified into three types based on their biogenesis and cellular roles: siRNAs, piRNAs, and miRNAs. miRNAs, the best understood of the three classes, have been shown to regulate at least 30% of human genes. Moreover, abnormal miRNA expression is linked to a variety of diseases, including neurodegenerative disorders, cardiovascular disease, and, most noticeably, human cancer. Furthermore, the expression of several miRNA species is altered during the progression of cancer. These specific-expression miRNAs have been identified as promising biomarkers in diagnostic procedures because of their clinical significance [21].
In a study, Xiaoyu Cai and co-workers developed and applied a microfluidic paper-based laser-induced fluorescence sensor based on duplex-specific nuclease (DSN) amplification to detect miRNAs in cancer cells in a selective and sensitive manner. DSN and Taqman probes were preserved in the circles of the folded paper chip under ideal conditions. When miRNA solution is added, the DSN can cyclically digest hybrids of miRNAs and Taqman probes, causing fluorescence signal amplification. Finally, the method was used to detect miRNA-21 and miRNA-31 in cancer cell lysates from A549, HeLa, and hepatocyte LO2. MiRNA-21 and miRNA-31 were detected in A549 and HeLa cells. Despite the amplification process, the analysis time was less than 40 min, and it detects very low levels of analytes [22].
Liang et al. present a fluorescent and visual method for multi-monitoring cancer cells in μPADs using GO-based aptameric biosensors. The design was based on the exceptional extinguishing capacity of GO in this method. Aptamers labeled with QD-coated mesoporous silica nanoparticles can be adsorbed on the surface of GO in a flexible single-stranded state, and the fluorescence is quenched via Förster resonance energy transfer, followed by fluorescence regeneration when target cells are added. The most important characteristic of the system was that it could be identified to multiplexed monitoring at the same excitation wavelength, making multiple detections much easier than methods using traditional organic dyes [23].
Huaping Deng et al. founded a portable and usable integrated paper fluid chip device that enables in situ HP-EXPAR amplification and optical sensing. It was created in a multi-layer format using simple paper folding. MiRNAs 155 and 21 were chosen as targets to test the performance of the platform, which used QDs as signal tags. At 90 min, the experiments yielded a satisfactory detection limit of 3 × 106 copies [24].
Neda Fakhri and colleagues worked on the design of a Y-shaped μPAD. They reported that the presented method is the first paper colorimetric miRNA-21 assay based on nanocluster catalytic activity. Due to the peroxidase mimetic activity of DNA-Ag/Pt NCs, this new paper-based biosensor was created to detect sub-micromolar concentrations of miRNA-21. The detection mechanism is based on the inhibitory effect of miRNA-21 on the activity of the formed nanocluster. The system was tested on a human urine sample and the colorimetric method proved to be sufficiently accurate [25].
In a different study, researchers developed a microfluidic paper-based fluorescent biosensor in which the T-shaped duplex structure was obtained by dynamically self-assembling for the quantitative detection of miRNA and folate receptor (FR). Layered MnO2 nanolayers exhibiting fluorophore quenching ability were synthesized. The synthesized construct and fluorophore-labeled ssDNAs were used in the biosensor design. This recently developed biosensor has a relatively low detection limit of 0.0033 fM and could detect miRNA-21 sensitively from 0.01 to 5.0 fM. Additionally, the current sensing system has a detection limit of 0.667 ng/mL and can detect FR in the range from 2.0 to 30.0 ng/mL [26].
Liquid biopsies are a non-invasive and easily applicable technique. However, analysis using different steps such as ultracentrifugation, immunomagnetic beads, or commercial kits requires time and money. Recently developed microfluidic paper-based platforms can effectively separate and detect biomarkers from liquid biopsies with higher sensitivity than conventional methods. They can also lead to detection in potential POC applications.
Table 1. Summary of microfluidic paper-based biosensor applications in the detection of cancer biomarkers using various analysis methods in the literature.
Analyte Cancer Types Bioreceptor Fabrication Technique Detection Technique/Signal Surface Chemistry Linear Range LOD Reference
CEA and NSE Lung CEA and NSE aptamers Wax printing and screen printing Label-free electrochemical detection/DPV Amino functional graphene (NG)–Thionin (THI)–gold nanoparticles (AuNPs) and Prussian blue (PB)–poly (3,4-ethylenedioxythiophene) (PEDOT)–AuNPs nanocomposites Linearity in ranges of 0.01–500 ng mL−1 for CEA (R2 = 0.989) and 0.05–500 ng mL−1 for NSE (R2 = 0.944), 2.0 pg mL−1 for CEA and 10 pg mL−1 for NSE. [5]
CEA, AFP, CA125, and CA19-9 Colorectal cancer Anti-CEA, AFP, CA125, and CA19-9 Antibody photolithography Colorimetric method/chemiluminescent Secondary antibody labeled with HRP, Luminol - 0.89 ng mL−1 for CEA,
1.72 ng mL−1 for AFP,
3.62 U mL−1 for CA125 and 1.05 U mL−1 for CA19-9		 [17]
CEA Lung and other Anti-CEA Antibody Wax printing and screen printing Label-free electrochemical detection/DPV NH2-G/Thi/AuNPs nanocomposites 50 pg mL−1 to 500 ng mL−1 10 pg mL−1 [8]
AFP Liver Anti-AFP antibodies Photolithography Labelled electrochemical detection/SWV rGO-TEPA/Au nanocomposite 0.01–100.0 ng mL−1 0.005 ng mL−1 [15]
EGFR Lung and other Anti-EGRF aptamer Wax printing and screen printing Label-free electrochemical detection/DPV NH2-GO/THI/AuNP nanocomposite 0.05 to 200 ngmL−1 (R2  =  0.989) 5.0 pgmL−1 [15]
NSE Lung Anti-AFP antibodies Wax printing and screen printing Wireless point-of-care testing (POCT) system with electrochemical/DPV NH2-G/Thi/AuNPs nanocomposite 1.0 ng mL−1 to 500 ng mL−1 10 pg mL−1 [10]
VEGF-C - Anti-VEGF antibody Wax printing and screen printing Label-free electrochemical detection/DPV and CV The NMB/SWCNT/AuNPs three-in-one nanocomplex 0.01–100 ng/mL 10 pg/mL [13]
PSA Prostate Anti-PSA aptamer Wax printing and screen printing Label-free electrochemical/DPV AuNPs/rGO/THI nanocomposites 0.05 to 200 ng mL−1 10 pg mL−1 [9]
CA 15-3 Breast Anti-CA 15-3 antibodies Inkjet printing /
electrodeposition		 Electrochemical detection/ChA Ag/RGO nano-ink and CysA/Au NPs 15–125 U/mL 15 U/mL [14]
CA 125 Ovarian Anti-CA 125 antibody Inkjet printing/electrodeposition Electrochemical detection/ChA Ag/RGO nano-ink and CysA/Au NPs 0.78–400 U/mL. 0.78 U/mL [27]
AFP and CEA Hepatocellular carcinoma and Colorectal Anti-CEA and AFP antibody layer-by-layer assembly and screen-printing Electrochemical detection/impedimetric - - 102 ng mL−1 [11]
Cancer cells of A549 and HeLa Lung and Cervical MiRNA-21 and miRNA-3 Wax printing Colorimetric method/laser-induced fluorescence Taqman probes - 0.20 ve 0.50 fM [22]
MCF-7, HL-60, and K562 cancer cells Breast and leukemia MSNs/QDs-labeled aptamers Wax patterning Colorimetric method/laser-induced fluorescence QDs, MSNs/QDs and MSNs/QDs–DNA 180 to 8 × 107, 210 to 7 × 107, 200 to 7 × 107cells mL−1 6270 and 65 cells mL−1 [23]
AFP Liver, ovaries, or testicles Primary
antibodies (Ab1) and secondary antibodies (Ab2)		 Wax patterning Colorimetric method with LIF detection/laser-induced fluorescence Hairpin strand-FAM 2.5–1000 pg/mL 1.0 pg/mL. [6]
Trx-1 Breast Anti-Trx-1 antibody-conjugated with HRP Stacking layers/cutting Colorimetric method/optic AgNP and Teflon ink 0–200 ng/mL - [7]
miRNA 155 (miR-155) and 21 (miR-21) - Nucleic Acid Sequences Wax and screen printing Colorimetric method/fluorescence QD-labeled probes, EXPAR template of miR-21, EXPAR template of miR-150. 3 × 105 to 3 × 108 copies 3 × 106 copies [24]
miRNA-21 (human urine sample) - ssDNA
template sequence		 Cutting Colorimetric method/optic DNA–Ag/Pt NCs 1.0–700 pM 0.6 pM. [28]
miRNA-21 and FR Breast Hairpin DNA Sequences Wax printing Colorimetric method/fluorescence Au nanoflowers (AuFLs) and MnO2 nanosheets For miRNA-21 0.01 to 5.0 fM
For FR
2.0 to 30 ng/mL		 For miRNA: 0.0033 fM; for FR:
0.667 ng/mL		 [26]
CEA - Anti-CEA antibodies Photoresist-coated Colorimetric method/chemiluminescent Fluorescein isothiocyanate (FITC)-labeled CEA antibody 1.0–80 ng mL−1 - [2]

This entry is adapted from the peer-reviewed paper 10.3390/bios13030387

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