Table 1.

Development of μPAD to detect different biomarkers (Blood-based, and Non-Blood-based) over the last decade.

DEVICE DESCRIPTION/ARCHITECTURE	SENSING MECHANISM	TARGET MOLECULE	ANALYTICAL PERFORMANCE	REAL SAMPLE	REFERENCE
Plasma separation μPAD •With the plasma separation zone in the center and four testing zones on the periphery •Hydrophobic barrier was printed using a wax printer onto a chromatography paper •The patterned paper was heated on a hot plate to melt the wax and to create the hydrophobic barrier spanning the entire thickness of the paper substrate.	Colorimetric Assay. Colour change of potassium iodide starch (from colorless to brown) in the presence of hydrogen peroxide was produced by oxidation of glucose by glucose oxidase.	Plasma glucose	NR	Venous blood	[6]
Plasma Separation μPAD •With a bottom separation zone and two top detection zones •Connected through a y-junction. •Patterned using wax dipping methods.	Plasma separation simultaneously with a colorimetric assay. Protein determination based on Bromocresol Green (BCG) method was performed.	Plasma Protein	LR 1.6–5.3 g dL-1	Blood	( [20]
Nitrite detecting μPAD (in saliva) •With the main channel with four identical arms and four testing zones •Designed using black lines and shapes on a white background •Printed with a wax toner onto grade 1 thin chromatography paper using a laser printer.	Colorimetric Assay. Griess reagent was used, which lead to the formation of a magenta-azo-compound whose color intensity relates to the concentration of nitrite	Nitrite	LR 10–1000 μmol/L LOD 10 μmol/L	Saliva	[40]
DNA detecting μPAD •A PDDA-GR (Poly-(Diallyldimethylaluminiumchloride)-functionalized Graphene) modified Au paper working electrode (PWE). •An Au paper working electrode modified by PDDA-GR and hybridized by incubation with a varying concentration of target DNA and then with reporter probes (S3–CaCo3/cma@ AgNPs bioconjugates).	Electro-chemiluminescence. Signals related to the target DNA (s2) concentrations were measured from the Paper Working Electrode (PWE).	DNA	LR 4.0 × 10-17 – 5.0 × 10-17 M LOD 8.5 × 10-18 M	Human Serum	[34]
A multiplex microfluidic (nitro-cellulose) paper-based immunoassay •With eight channels and a central zone. •Patterned by Craft Punch Patterning (CPP) in a die-cast mechanism under ambient temperature.	Antigen-Antibody reaction. Based on the quantitative detection of mouse IgG, qualitative detection of anti HCV by using an equal mixture of individual recombinant HCV antigens, including Core, NS4, and NS5 (except NS3) was demonstrated.	Anti-HCV (Core, NS5, AND NS4)	LOQ ∼40 pg LOD ∼1 pg	Human Serum	[23]
UTI biomarker detecting μPAD •With eight distribution channels and eight square zones with a dimension of 5 mm at the ends of the channel with a width of 3 mm. •Printed on a PMMA plate •Patterned using CO2 laser engraving method	Colorimetric Assay. Sample reacted with 50 mm sulfanilamide,10 mm n-(1-naphthyl)-ethylenediamine 330 mM citric acid in methanol to produce a red-violet color	Urinary Tract Infection biomarkers. (NITRITE)	NR	Urine	[24]
A handheld,eight-channel potentiostat •With an E-μPIA in its electrical connection slot. •The eight reaction zones were patterned on a piece of cellulose chromatography paper via wax printing •Electrodes were formed on the reaction zones via stencil printing	Indirect ELISA. Indirect ELISA of antibodies to HIV p24 and HCV core antigens were realized on an E-μPIA handheld potentiostat with a Bluetooth module that was developed for readout of output signals from the device.	HIV AND HCV antibodies	LOD 300 pg/mL, for HIV 750 pg/mL, for HCV	Mouse Serum	[24]
μPAD for sensitive electrochemical detection of carcinoembryonic antigen	Electrochemical Sensing	Carcinogenic agents	LR 50–500 pg/mL LOD 10 pg/mL	Human Serum	[6]
Glucose detecting μPAD •With eight zones interconnected by microfluidic channels with a central zone for the sample inlet •Fabricated by a stamping process.	Colorimetric Assay. Performed with the help of a selected chromogenic agent (4-AAP/DHBS, KI and TMB FOR glucose detection and 4-AAP/DHBS and OD for uric acid) with an office scanner using 600 dpi resolution. The recorded images were converted to the RGB scale and analyzed.	Glucose and Uric Acid	LOD: 23 μM, for Glucose 37 μM, for Uric Acid	Artificial Serum Sample Human Tear Sample	[45]
Ketamine detecting E-μPAD •Integrated with Nano-crystal Zeo-GO •The working and counter electrode was ornated on the paper using commercial conducting carbon ink •Hydrophilic channels with hydrophobic barrier were printed using wax printing followed by heating	Electrochemical Sensing. Electro-oxidation of drugs amplified by the use of Nano-crystalline Zeo-GO (Zeolite-graphene oxide)	Ketamine (Anaesthetic)	LR 0.001–5 nM/mL DL 0.001nM/mL	Ketamine samples	[64]
Blood Iron analyzing μPAD •Made by masking filter paper by PDMS which bonded by plasma bonding •Followed by hydrothermal deposition of Carbon Dots (CD) followed by Au-NP plating. •Two regions were formed –one with Au-NPs on NF-CDs and the other with F-CDs	For Fe3+: 5 ml of ascorbic acid was added to 1 ml OF human blood sample mixed with 2 mL of red blood cell lysate buffer and incubated at room temperature for 5mins. A fluorescence quenching test was done on the supernatant of this mixture. For Ferritin: Colorimetric ELISA For Ferritin Analysis On AuNPS/NF-CDs region was done.	Fe3+ and Ferritin	NR	Blood	[21]
The μ-PAD had a pattern •Constituted of a channel with a larger area on top for loading of sample and four small areas all separated by lines of yellow wax. •The pattern was wax printed.	Antigen-Antibody Reaction. P122 and P82 polyclonal antibodies were used to detect specific antigens.	Dengue and Zika NS1 Detection	LOD (whole blood) 20 ng/mL, for Dengue and ZIKV	Whole Blood	[25]
NSE detecting μPAD •With two circular zones –one sample zone with a diameter of 3 mm and the other one is for an auxiliary zone with a diameter of 3.5 mm adopting three electrodes system (working, reference and counter electrodes) •Printed by wax printing and screen printing techniques.	Electrochemical Sensing	Neuron Specific Enolase	LR 1–500 ng/mL LOD 10 pg/mL	Serum	[36]
Creatinine detecting μpad •Consisting of the main channel and three identical arms each containing a circular testing zone •Circular uptake zone printed using a wax printer	FOR CREATININE: 2 μL aliquot of a solution of 0.1 mol/l picric acid in 1 mol/l NaOH was spotted onto the uptake zone. For Uric Acid 2 μL aliquot of a solution containing 1,10-phenanthroline(0.06 mol/l)and acetate (0.53 mol/l)was added to the uptake zone and a 2 μl aliquot of a solution of fe3+(0.45 mol/l)in h2so4(0.25mol/l)was spotted onto the testing zone	Creatinine Uric Acid	LR 50–600 mg/L, for Creatinine 50–500 mg/L, for Uric Acid LOD 15.7 mg/L, for Creatinine 16.5 mg/L, for Uric Acid	Urine	[43]
A paper plastic microchip (PPMC) •Comprising of three-layer hybrid substrates prepared of a cellulose paper substrate assembled with a transparent plastic sheet by double-sided adhesive •Laser cutting was used for patterning •Screen printing to print electrodes on the surface of the paper.	FOR ZIKV DETECTION, Viral lysate prepared using a 1% Tritonx-100 solution was used after capturing the ZIKV particles using magnetic beads modified with anti-ZIKV envelope monoclonal antibody(anti-ZIKV mab), followed by impedance testing with different virus concentrations. (with the increase in viral concentrations, the impedance decreased; which could be attributed to the increase in charged particles released during the lysis step. FOR HPV, Loop-mediated isothermal amplification was used, which resulted in the loop like amplicons for both HPV16 and 18. FOR MULTIPLEXED DETECTION OF ALPHA FETOPROTEIN AND CARCINOEMBRYONIC ANTIGEN (CEA). PPMC designed with two semi-circular electrodes and two detection zones modified with biomarker specific monoclonal antibodies were used (the increase in concentrations resulted in a decrease in impedance)	Zika Virus, HPV, Alpha-Fetoprotein And Carcinoembryonic Antigen (CEA)	LOD For cancer protein biomarkers, 100 ng/mL For viral particles, 1000 particle/mL For HPV nucleic acid, 100 copies/mL	Serum	[37]
A μPAD for multiplex detection of cardiac biomarkers with three reaction zones and one sample zone at the center.	Colorimetric detection of multiplex cardiac markers by using only an available phone camera or desktop scanner for quantification. For GPBB detection. The target analyte was sandwiched between a capturing antibody and a detecting antibody and color signals were observed from the labeled nanoparticles conjugate –detecting antibody. (yellow for GPBB, purple for CK-MB and red for cTnT) For a particular analyte. Three different colored NPS (gold, silver and gold urchin) were used as detecting indicators.	GPBB (Glycogen Phosphorylase Isoenzyme BB) with CK-MB (Creatine Kinase-MB)And cTnT Cardiac Troponin T	LOD For GPBB, 0.5 ng/mL For CK-MB, 0.5 ng/mL For cTnT, 0.05 ng/mL	SERUM	[28]
GO-modified μpads coupled with smartphone-based colorimetric detection for direct quantification Fabricated by methods of photolithography	Graphene Oxide enhanced colorimetric assay	Glucose	LOD 0.02 mM LR 0∼1 mM	Artificial Saliva	[46]
Paper-based electrochemical sensing platform μPEC for Thrombin detection •The hydrophobic barriers were fabricated through wax printing •The Ag/AgCl reference electrode and a carbon counter electrode were obtained through screen printing.	Paper-based TiO2 Nano-sheets (PTNs) and CeO2 were employed as an electron transporting material and photoactive material respectively. Upon illumination, the photogenerated electrons of CeO2 promptly transferred into PTNs increasing in photocurrent intensity. By regulation of electron-transfer tunneling distance between PTNs and CeO2 sensitive detection of Thrombin was realized.	Thrombin	LR 0.02pM- 100 pM DL 6.7 fM	Human serum	[27]

NR - not reported | LOD – Limit OF Detection | LR – Linear Range | LOQ – Limit Of Quantification | DL – Detection Limit.