Folic acid biosensors: A review

Process Biochemistry xxx (xxxx) xxx–xxx

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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Folic acid biosensors: A review Bhawna Batraa, Vinay Narwalb, Vijay Kalrac, Minakshi Sharmad, JS Ranaa,* a

Department of Biotechnology, Deen Bandhu Chhotu Ram University of Science and Technology, Sonipat, India Department of Biochemistry, M.D. University, Rohtak, India Department of Biochemistry, University of Health Sciences, Rohtak, India d Department of Zoology, M.D. University, Rohtak, India b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Folic acid (FA) FA determination methods FA biosensors Nanomaterials Biological samples

Folic acid (FA) also known as (N-[p-{[(2-amino-4-hydroxy-6-pteridinyl) methyl] amino} benzoyl]-L-glutamic acid), is a water soluble vitamin found in plants and animals. The deficiency of FA leads to an increased risk of various diseases like neural tube defects in newborn, cardiovascular disease, cancer, Alzheimer’s disease and megaloblastic anemia. The normal levels of FA in human blood serum should fall in the range between 2 and 15 ng/mL. Present review article discusses the classification, principles, advantages and disadvantages of FA biosensing methods. FA biosensors operate within 3–300 s, in pH range, 1.8–7.8, concentration range 8.71 × 10−9 μM for FA. The FA biosensors displayed detection limits (LOD) between 1.6 × 10−11 to 0.091 μM and with working potential −0.88 to 4.5 V. These biosensors measured FA level in various biological and pharmaceuticals samples.

1. Introduction Folic acid (FA), a water soluble vitamin also known as vitamin M is responsible for synthesis of red blood cells. The common name of FA is vitamin B9 [1]. Its natural form is known as folate. FA molecule consists of bicyclic pterin moiety joined by a methylene bridge C(9)-N(10) to pamino benzoic acid, which in turn is connected to L-glutamic acid with the help of peptide bond. In various food sources, FA is found in its reduced forms i.e 7, 8- dihydrofolate (DHF) or 5,6,7,8- tetrahydrofolate (THF) [2]. FA is essential for various human metabolic activities. It is a major factor in the nucleic acid synthesis [3]. It accelerates the cell division and therefore helps in growth and development of foetus [4]. In human beings, folate level is found about 10 mg–30 mg [5]. The normal level of folate in serum is about 5–15 ng/mL, while in cerebrospinal fluid it is about 16–21 ng/mL [6]. In erythrocytes it is found in the range from 175 to 316 ng/mL [7]. Liver consists of abundant amount of folate as compared to the blood and various tissues. The range below 5 ng/mL of serum folate indicate the symptoms of its deficiency, which causes a large number of disorders [8]. The folate contents present in food are primarily hydrolyzed by the intestines and converted to monoglutamate form, which is actively absorbed by small intestine mucosa whereas FA, when consumed as a supplement is absorbed passively by the proximal portion of the small intestine [9]. Moreover, monoglutamate is further reduced to THF in the liver and

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converted to either the methyl or the formyl forms before entering the bloodstream [10]. Therefore, in blood folate is found in the form of 5methyl-tetrahydrofolate. Defect in 5-methyl-tetrahydrofolate causes hyperhomocysteinemia or high plasma homocysteine at a level higher than16 μmol/L and lower values of 12–14 micromoles/L [11]. The FA metabolic products appear in the urine after 6 h of their ingestion and complete excretion is generally within 24 h [12]. Folate is also used in repairing and synthesis of methylate DNA especially during pregnancy. Its deficiency can lead various health problems [13]. Hyperhomocysteinemia and cancer development due to impaired DNA synthesis and repair could be the long-term complications of folate deficiencies [14]. These concerns have led the researchers to design various analytical devices to detect FA such as spectrophotometry [15], high performance liquid chromatography (HPLC) [16], colorimetric [17], flow injection chemiluminescence [18] and fluorimetric [19]. However, these methods possess some major drawbacks like high cost instrumental setup, expertise in operating the instruments, sample pre-treatment and time consuming procedure. To overcome the drawbacks of traditional methods, biosensing methods are designed for FA monitoring. These methods are found highly specific, sensitive, cheap, fast and reproducible [20,21]. Recently, to improve the analytical performance and electron kinetics of electrochemical biosensors, nanomaterials have been incorporated in designing process of biosensors [22]. A number of electrochemical FA biosensors have been reported such as carbon fibre

Corresponding author. E-mail address: [email protected] (J. Rana).

https://doi.org/10.1016/j.procbio.2020.01.025 Received 25 September 2019; Received in revised form 4 January 2020; Accepted 22 January 2020 1359-5113/ © 2020 Published by Elsevier Ltd.

Please cite this article as: Bhawna Batra, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.01.025

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been developed either without or with nanomaterials. Sensors without incorporating nanomaterials have displayed poor sensitivity, less electron communication, narrow linearity, more response time, and less stability. To mitigate all these pitfalls, nanomaterials have been exploited for fabrication of FA biosensors. They have drawed the attention, due to their small size, high surface area, electrocatalytic and optical features including high electron conductivity and sensitive nature [61,62]. Moreover, these devices are highly economic and fast.

microelectrode based FA biosensor [23], mercury meniscus modified silver solid amalgam electrode based FA biosensor [24], molecular imprinted polymer based FA biosensor [25,26], 2-mercaptobenzothiazole self-assembled Au electrode based FA biosensor [27], α-polyoxometalate–polypyrrole–Au nanoparticles modified gold electrode [28], AuNPs modified Au electrode based FA biosensor [29], reduced graphene modified Au electrode based Folic biosensor [30], ordered mesoporous carbon based FA biosensor [31], phosphomolybdic-polypyrrole film modified glassy carbon electrode based FA biosensor [32], thiadiazole modified glassy carbon electrode based FA biosensor [33], bismuth nanowires modified glassy carbon electrode based biosensor [34], single-wall carbon nano-tube (SWCNT) film modified glassy carbon electrode based FA sensor [35], single-walled carbon nanotubeionic liquid paste coated glassy carbon electrode based FA biosensor [36], copper oxide nanoleaves and multiwalled carbon nanotubes modified glassy carbon electrode based FA biosensor [37], hydroxyapatite nanoparticles modified glassy carbon electrode based Folic acid biosensor [38], phosphomolybdic acid–polypyrrole/graphene composite modified glassy carbon electrode [39], platinum nanoparticles doped multi-walled carbon nanotubes modified glassy carbon electrode [40], MoS2/reduced graphene oxide modified glassy carbon electrode [41], nanostructured α-Fe2O3 based glassy carbon electrode [42], Cu doped SnO2 nanoparticles modified glassy carbon electrode [43], nickel ions dispersed poly(o-anisidine) film modified carbon paste electrode based FA biosensor [44], sodium alpha olefin sulphonate modified carbon paste electrode based sensor [45], calixarene chemically modified electrodes based FA biosensor [46], boron doped diamond electrode based FA sensor [47], mercury film electrode based FA sensor [48], sol-Gel carbon ceramic electrode based FA biosensor [49], lead film electrode based FA sensor [50], poly(brilliant cresyl blue)multiwall carbon nanotube composite film modified indium tin oxide (ITO) electrode based FA biosensor [51], DNA modified-pencil graphite electrode (PGE) based FA biosensor [52], immunoassay based on electrochemical magneto sensor for FA detection [53], BSA-modified gold nanoclusters based fluorescence biosensor for FA detection [54], enzyme linked immunosorbent based FA biosensor [55], immunoaffinity based optical biosensor for FA detection [56], folate binding protein based optical FA biosensor [57], carbon nanohorns supported interwoven titanate nanotubes based photo electrochemical sensor for FA detection [58], ZnO nanowires array based FA biosensor [59]. This article provides a comprehensive review for the various FA biosensors, their classification, principles, merits and demerits. Future perspectives have also been discussed to improve the biosensing technology in terms of cost and real time monitoring.

3. Classification of electrochemical Folic acid biosensors The electrochemical FA biosensors are classified according to their electrodes such as carbon fibre microelectrode, mercury meniscus modified silver solid amalgam electrode, molecular imprinted polymer (MIP), gold electrode (AuE), glassy carbon electrode (GCE), carbon paste electrode (CPE), boron doped diamond electrode, mercury film electrode, lead film electrode, optical biosensor and carbon nanohorns supported interwoven titanate nanotubes electrode. 3.1. Carbon fibre microelectrode based Folic acid biosensor An electrochemical biosensor based on carbon fibre microelectrode was designed using pretreatment regime and optimized for the determination of FA. The functionalized electrode exhibited higher faradic and capacitive currents, due to the modification of surface. To check the analytical performance of functionalized electrode, differential pulse voltammograms (DPV) of FA observed in 0.1 M perchloric acid. The electrode showed well defined reduction peak having a LOD of 1 × 10−8 M with a wide linear range of 2 × 10−8 M to 1 × 10−6 M FA [23]. Merits: Pre-treated carbon fibre microelectrode has been considered as a promising tool in determination of FA due to their small size, sensitivity, good electrochemical properties. Demerits: Carbon fibre electrodes are relatively expensive and difficult to machine. Moreover, they are prone to mechanical damage during the use. 3.2. Mercury meniscus modified silver solid amalgam electrode based Folic acid biosensor A new approach has been developed using mercury meniscus modified silver solid amalgam electrode (m-AgSAE) to detect the FA. The FA behaviour was investigated using cyclic voltammetry (CV), DPV and adsorptive stripping differential pulse voltammetry (ASDPV). The detection was carried out in a 1:9 mixture of methanol and aqueous acetate buffer. The electrode showed the LOD 0.5 nmol/L and RSD less than 4 % [24]. Merits: m-AgSAE presents good relative advantages i.e the long time stability, simple electrochemical activation, and easy renewal of mercury meniscus, which makes this electrode as a suitable choice for FA monitoring. Demerits: m-AgSAE shows limited anodic window due to mercury oxidation. Furthermore, the mercury present in electrode exhibits toxicity towards the biological samples.

2. Concept of biosensor Biosensor is an electroanalytical device used to monitor the specific analyte present in biological samples (Fig. 1) [60]. The basic principle of FA biosensor is to produce an electrical signal which is proportional to the quantity of monitored FA. As FA biosensor is used in detection of various diseases like cancer, cardiovascular diseases, neural tube defects and megaloblastic anaemia, therefore, it must be a hot topic in biosensing industries. Numerous techniques have been developed in designing of FA biosensors. Though a number of sensors for FA have

3.3. Molecular imprinted polymer based Folic acid biosensor MIP based FA sensor was designed using the conducting carbon particles which were arranged in a order form on the carbon strip using the technique free radical polymerisation with a new monomer (2,4,6trisacrylamido-1,3,5-triazine, TAT). The carbon strip was inducted in the polymer for direct electronic conduction (Fig. 2). FA molecules which were exclusively entrapped in MIP cavity, got oxidized on the surface of MIP-fibre sensor. Cyclic voltammogram showed well defined redox peaks as shown in supplementary Fig. 1. The method was found more specific and exhibited LOD of 0.20 ng/ml with RSD = 1.3 %. The

Fig. 1. Basic principle of biosensor [60]. 2

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Fig. 2. Systematic presentation of the synthesis of MIP–carbon composite [25].

3.4. Au electrode based Folic acid biosensor

analysis was carried out in blood serum and pharmaceutical samples. The interference was found negligible during detection of FA in biological samples [25]. An another MIP based biosensor was designed using pre anodized sol–gel coated pencil graphite (PG) (grade 2B) electrode (Fig. 3). The MIP was incorporated on the exterior surface of PG. The SEM image exhibited homogeneously dispersed colloidal particles having enhanced surface area (Supplementary Fig. 2). The electrode was preconcentrated at +0.8 V (with respect to Ag/AgCl). The FA molecules, entrapped in MIP cavity, got oxidized irreversibly at C9-C10. The broader CV peak (at −0.55 vs Ag/AgCl at 10 mV/s) showed reduction behaviour of oxidized FA in the MIP cavity. The encapsulated analyte was oxidized and cathodically stripped off using differential pulse cathodic stripping voltammetric signal. The FA biosensor showed LOD of 0.002 μg/mL (3σ, RSD ≤ 3.0 %), without any interference and complications [26]. Merits: MIPs can be developed for any template. They are stable in the harsh conditions (pH, temperature, ionic strength, solvents). They exhibit long term storage without loss in performance. They can satisfy the needs of simple, fast, cost-effective and robust biosensors. Demerits: MIPs do not perform well in the presence of water which can be found in biological or environmental samples. The polarity of water can interfere with interactions between the target analyte and the imprinted sites. This would have a greater effect if specificity relied on non-covalent interactions such as hydrogen bonds but would be less of a problem for covalent imprinted polymers.

3.4.1. 2-mercaptobenzothiazole self-assembled Au electrode based Folic acid biosensor The 2-mercaptobenzothiazole self-assembled Au electrode (MBT/ SAM/Au) based biosensor was designed. FA bound strongly to MBT/ SAM/Au and form a tightly packed monolayer. The chronocoulometry demonstrated the surface adsorption of folic acid at the MBT/SAM/Au electrode. Reproducible voltammogram was obtained when MBT/SAM/ Au electrode was dipped into folic acid solution (Supplementary Fig. 3). The average electron transfer rate of electrode was predicted as 0.085 s−1 with a maximum surface adsorption 2.8 × 10−10 mol cm−2 having adsorption equilibrium constant of (4.0 ± 0.2) × 105 l mol−1, and an electron charge resistance of 12,489 Ω. The oxidation currents achieved on electrode show linear response with the logarithm of the content of FA in the range of 8.0 × 10−9–1.0 × 10−6 mol l−1. The LOD was 4.0 × 10−9 mol l−1 [27]. Merits: 2-mercaptobenzothiazole self-assembled gold electrode exhibited excellent electron kinetics, high sensitivity and high surface area. Demerits: As monolayer was physically adsorbed on the surface of Au electrode, it may leach out and lead to less stability. 3.4.2. α-polyoxometalate–polypyrrole–Au nanoparticles modified gold electrode In the study, gold nanoparticles (AuNPs) and α-polyoxometalate (αPOM) (K7PMO2W9O39·H2O) were simultaneously encapsulated into electropolymerized polypyrrole (PPy) film using the CV technique 3

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Fig. 3. Schematic representation of the preparation of the MIP–sol–gel coated PGE [26].

(Supplementary Fig. 4 B). The modified electrode showed excellent electron kinetics at 0.3 V (vs. SCE) with the electron transfer rate constant (ks) of 1.15 × 10−19 s−1 [28]. Merits: α-polyoxometalate–polypyrrole have uniformly arranged well defined molecular complexes and have high electrocatalytic activity. Demerits: Study was not co-related with standard method available for folic acid determination.

(Fig. 4). The SEM images revealed a porous like structure composite film nanometer- sized particles (250 nm) due to the successful electropolymerization which produces excellent electrochemically for reduction of FA (Supplementary Fig. 4 A). The PPy–α-POM–AuNPs modified Au electrode was examined using square-wave voltammetry (SWV). The cyclic voltammogram showed the electrochemical behaviour of FA on the PPy–α-POM–AuNPs modified Au electrode. α-POM & AuNPs synergistically contribute a good conductive electron pathway for large transfer of electrons from the electrode-elctrolyte interface

Fig. 4. Schematic representation for the fabrication of PPy–α-POM–AuNPs film modified Au electrode [28]. 4

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provides the development of highly sensitive and selective biosensor for FA detection. Demerits: It is tedious to prepare. It has not been applied for real samples.

3.4.3. AuNPs modified Au electrode based Folic acid biosensor The AuNPs decorated Au electrode based FA biosensor has been designed. The surface of the modified gold plate before and after activation was scanned by scanning electron microscopy (SEM) (Supplementary Fig. 5). FA molecules got adsorbed on the surface of AuNPs modified gold electrode & exhibited cyclic voltammogram. The biosensor worked optimally at a scan rate of 100 mV/s with an applied potential of 4.5 V. The biosensor provided a rapid response of about 1 min. Moreover, a good linear response was obtained in the range of 1.0 × 10−8 to 1.0 × 10−6 mol L−1 with LOD of 7.50 × 10−9 mol L−1. The method evidenced high specificity and selectivity for FA. The biosensor was used in real samples such as FA medicines, flours, and vegetables [29]. Merits: AuNPs offers significant advantages in fabrication of biosensor due to (i) simple synthesis and fabrication process and (ii) high biocompatibility with living tissues. Demerits: This is not specific for the folic acid as at high potential number of analytes get oxidized. It showed less stability.

3.5.3. Thiadiazole modified glassy carbon electrode based Folic acid biosensor A novel biosensor was constructed for the estimation of FA in presence of ascorbic acid (AA) and uric acid (UA). The biosensor was constructed using 5-amino-2-mercapto-1,3,4-thiadiazole (p-AMT) modified GCE. The modified electrode showed different voltammetric response for AA, UA and FA but offered high oxidation peak current for these analytes. FA molecules came in contact with the surface of p-AMT film modified GC electrode & got oxidized as shown by oxidation peak in cyclic voltammogram (Fig. 8). The electrode showed high sensitivity and selectivity for the FA detection. The LOD of biosensor was 75 nM for UA and 100 nM for both AA and FA. Moreover, the biosensor displayed high concentration range of 1.0 × 10−7–8.0 × 10−4 M for FA. The electrodes were applied in detection of analytes in human serum sample [33]. Merits: Thiadiazole moieties are promising agents for fabrication of biosensor due to their excellent antifungal and high biocompatibility properties. Demerits: Accuracy of biosensor was not studied as it was not corelated with standard method.

3.4.4. Reduced graphene modified Au electrode based Folic biosensor A highly sensitive biosensor was fabricated for the estimation of FA using using electrophoretic deposition (EPD) of reduced graphene oxide (rGO) onto a gold electrode and it also involved the post-functionalization of rGO with FA. DPV showed a significant decrease in current upon FA binding. Cyclic voltammetry showed the electrochemical behaviour of FA onto the Au/rGO using three different redox probes. The sensor showed LOD of 1 pM. The method was applied in clinical samples for the detection of FA [30]. Merits: rGO has triggered analytical performance of FA biosensor owing to its high electroactive surface area and effective covalent bonding due to presence of large number of functional groups. Demerits: The gold electrode exhibits high cost, and time consuming procedure of immobilization of enzyme on it. Moreover, they exhibit larger cathodic potential range and anodic window is limited by surface oxidation.

3.5.4. Bismuth nanowires modified glassy carbon electrode based biosensor Bismuth nanowires (BiNWs) based biosensor was designed for estimation of FA content in pharmaceutical tablets. FE-SEM images of electrodeposited BiNWs modified GC revealed bundles of compact nanowires of bismuth (BiNWs) in a size range of 30 nm–70 nm (Supplementary Fig. 9 A & B). Cyclic voltammogram showed electrocatalytic nature of BiNWs toward FA reduction which is due to high surface to volume ratio facilitating fast electron transfer kinetics of FA electero-reduction (Supplementary Fig. 9C). The working electrode exhibited a linear response in concentration ranging 1 × 10−8–15 × 10−8 mol L−1 FA. The LOD displayed by biosensor was 31.68 × 10−9 mol L−1. Moreover, the biosensor also offered good coefficient of variation i.e. 0.997. Interfering components have negligible effect on the biosensor response [34]. Merits: Bi nanowires should considered as effective Nanomaterials due to their high thermoelectric properties. Demerits: Though interference study was performed, yet it is nonspecific method for determination of folic acid.

3.5. Glassy Carbon Electrode based Folic acid biosensor 3.5.1. Ordered mesoporous carbon based Folic acid biosensor An ordered mesoporous carbon (OMC) based FA biosensor has been designed using GCE. TEM. displayed highly ordered carbon nanowires with mesopore range of 4–5 nm (Supplementary Fig. 6 A, B). It also exhibited the hexagonal honeycomb-like arrangement of mesopores. Cyclic voltammogram depicted the electrical oxidation of folic acid om the surface of OMC/GC electrode (Supplementary Fig. 6 C). The DPV measurements showed a working range from 5.0 × 10−10 to 1.0 × 10−7 M with a lower LOD of 6.0 × 10−11 M (S/N = 3) [31]. Merits: OMC is a flexible material providing interconnected channels for the diffusion of electroactive species in electrochemical systems. Demerits: OMC was physically adsorbed on the surface of GCE, it may leach out.

3.5.5. Single-wall carbon nano-tube (SWCNT) film modified glassy carbon electrode based Folic acid sensor SWCNT’s were modified onto GCE to fabricate a highly sensitive biosensor for determination of FA. FA molecules got electrically oxidized on the surface of SWNTs modified GC electrode. The pH effect was studied in the pH range from 4.0–8 with Britton-Robinson buffer solution. The peak current provided a linear response of FA concentration range from 1 × 10−8 to 1 × 10−4 mol L−1 with LOD of 1 × 10−9 mol L−1 after 5 min accumulation [35]. Merits: The film electrode provides an efficient way for eliminating interferences from some inorganic and organic species in the solution. The high sensitivity, selectivity and stability of the film electrode demonstrate its practical application from a simple and rapid determination of FA in tablets Demerits: It was not applied for determination of folic acids in real samples.

3.5.2. Phosphomolybdic-polypyrrole film modified glassy carbon electrode based Folic acid biosensor Keggin-type phosphomolybdate (PMo12) doped polypyrrole (PPy) film modified GCE (PMo12-PPy/GCE) was fabricated to sense the FA. The working electrode was fabricated using polymerisation technique. The oxidation and reduction peaks of electrodes were studied using Cv methods. Cyclic voltammogram showed oxidation of folic acid on the surface of PMo12-PPy/GC electrode in Supplementary Fig. 7. The working electrode was optimized in terms of pH, temperature and scan rate. The working electrode offered wide detection range of FA concentration from 1.0 × 10−8 to 1 × 10−7 M with LOD of 1.0 × 10−10 M [32]. Merits: Polypyrrole films exhibited high redox stability and thus

3.5.6. Single-walled carbon nanotube -ionic liquid paste coated glassy carbon electrode based Folic acid biosensor A single-walled carbon nanotube (SWNT) paste coated GCE has been prepared by using room temperature ionic liquid (i.e. 1-octyl-35

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Demerits: The working electrode was prepared by drop casting method which is not more durable.

methylimidazolium hexafluorophosphate, OMIMPF6) as a binder. SEM of SWNT film displayed highly dispersed (Supplementary Fig. 10 a, b). When OMIMPF6 was introduced it formed uniform black paste. The surface of the paste film was even and the SWNTs became indiscernible. The electrode (i.e. OMIMPF6–SWNT/GCE) combined the features of carbon nanotube and room temperature ionic liquid. FA could exhibit a very sensitive anodic peak at 0.75 V (vs SCE) in pH 5.5 phosphate buffer solutions, resulting from the irreversible oxidation of FA in supplementary Fig. 10 c. Under the optimized conditions, the peak current was linear to FA concentration over the range of 2.0 × 10−9 to 4.0 × 10−6 M by DPV, and the LOD was 1.0 × 10−9 M (S/N = 3). The OMIMPF6–SWNT/GCE was successfully applied to the determination of FA in real samples [36]. Merits: Small fractions of nanotubes improve the electrocatalytical efficiency by increasing the electron collection efficiency. Demerits: Paste of SWNT & ionic liquid (OMIMPF6) was just physically adsorbed on the surface of GCE, thus would lead to lesser durability/stability.

3.5.9. Phosphomolybdic acid–polypyrrole/graphene composite modified glassy carbon electrode A novel phosphomolybdic acid–polypyrrole/graphene composite modified GCE has been fabricated for the sensitive determination of FA based on the inhibitory activity of FA towards the redox behaviour of Keggin-type phosphomolybdic acid. TEM image of the GR exhibited a wrinkle-like thin sheet, characteristic of GR (Supplementary Fig. 13 a). SEM revealed structure of PMo12-PPy & PMo12-PPy/GR and found PMo12-PPy with an average size of ∼50 nm unevenly deposited on the glassy electrode. While PMo12-PPy were uniformly deposited on GR modified electrode with a porous three-dimensional structure as depicted by SEM (Supplementary Fig. 13 b & c). The modified GCE for its redox nature was investigated and studied using CV and DPV. It exhibited good linearity with FA concentration in the range from 1.0 × 10−9 to 2.0 × 10−7 M, and detection limit was found 3.3 × 10−11 M [39]. Merits: Phosphomolybdic acid-polypyrrole/GO modified GCE has high electrocatalytic activity, surface-controlled electron transfer process. The use of GO not only increases the electroactive surface area, but also facilitates electron transfer due to its high electric conductivity. Demerits: Deposition of phosphomolybdic acid–polypyrrole/graphene on the surface of was not supported by other studies such as FTIR.

3.5.7. Copper oxide nanoleaves and multiwalled carbon nanotubes modified glassy carbon electrode based Folic acid biosensor Present work described the formation of CuO nanoleaves using alcohol as reducing agent for reduction of Cu(II) chloride in the presence of poly(diallyldimethylammonium chloride) (PDDA) to determine FA. PDDA favoured polyol to develop CuO with well-defined leaf-like structure. They were characterized by Field emission scanning electron microscope (FESEM), Fourier-transform infrared (FT-IR) spectroscopy and X-ray diffraction (XRD) analysis. FESEM image confirmed the synthesis of CuO having leaf-like morphology and branched side edges. As prepared CuO nanoleaves was calculated to be 400 nm in length and 150 nm in width. The CuONs/MWCNTs/GCE nanocomposite modified electrode exhibited good electrochemical and electrocatalytic activity toward the oxidation of FA (Supplementary Fig. 11) with high sensitivity as 3.35 μA/μM and LOD (3σ) of 15.2 nM (S/N = 3). Besides, the CuO nanocomposite modified electrode lowered the over potential of FA oxidation than the unmodified electrodes [37]. Merits: CuO nanoleaves composite-modified electrode featured exhibit high sensitivity, excellent stability and fast amperometric sensing of FA. Demerits: As nanocomposite was physical adsorbed on the surface of electrode, it may leach out.

3.5.10. Platinum nanoparticles doped multi-walled carbon nanotubes modified glassy carbon electrode For electrochemical sensing of FA, platinum nanoparticles (PtNPs) in association with multi-walled carbon nanotubes (MWCNTs) with Nafion as the adhesives were used. PtNPs doped MWCNTs (PtNPs/ MWCNTs), were characterized by transmission electron microscopy (TEM) and electrochemical method, which revealed its good electrocatalytic activity for FA sensing. TEM showed the dispersion of MWCNTs, PtNPs & PtNPs/MWCNTs in Nafion matrix and found most of the MWCNTs in the form of small bundles with 25–40 nm in diameter (Supplementary Fig. 14). Synergistic effect of PtNPs & MWCNTs showed excellent electrocatalytic activity towards the oxidation of FA as shown in Supplementary Fig. 14. It exhibited a good linear relationship between the anodic peak current and FA concentration in the range 2.0 × 10−7–1.0 × 10−4 M. Detection limit was found 5.01 × 10−8 M having correlation coefficient R = 0.9948. The relative standard derivation was 1.9 % for 5 × 10−6 M FA in 11 repeated determinations [40]. Merits: PtNPs exhibit easy preparation methods, high flexibility and cost effectiveness. Moreover, the PtNPs modified electrode showed excellent sensitivity and stability for the determination of FA. Demerits: Nanocomposite of PtNPs and MWCNTs were physical adsorbed on the surface of glassy carbon electrode, it may detach and lead to less stability.

3.5.8. Hydroxyapatite nanoparticles modified glassy carbon electrode based Folic acid biosensor Present work reported the synthesis of hydroxyapatite (HA) nanoparticles (NPs) using a simple microwave irradiation method and employed for the development of FA sensing by electrochemical method. HANPs were characterized using XRD, FTIR, Raman and XPS. SEM and EDX studies confirmed the formation of elongated spherical shaped HANPs with an average particle size of about 34 nm (Supplementary Fig. 12 A & B). The HANPs thin film on glassy GCE was deposited by drop casting method. Electrocatalytic behaviour of FA in the physiological pH 7.0 was investigated by cyclic CV, LSV and chronoamperometry. Cyclic voltammogram (Supplementary Fig. 12) depicted the oxidation of FA on the surface of GC electrode modified with HANPs. The fabricated HA/GCE exhibited a linear calibration plot over a wide FA concentration ranging from 1.0 × 10−7 to 3.5 × 10−4 M with the LOD of 75 nM. In addition, the HANPs modified GCE showed good selectivity toward the determination of FA even in the presence of a 100-fold excess of AA and 1000-fold excess of other common interferents. The fabricated biosensor exhibited good sensitivity and stability, and was successfully applied for the determination of FA in pharmaceutical samples [38]. Merits: Hydroxyapatite nanoparticles are attracting interest in biosensing application due to its exceptional properties like biocompatibility, bioactivity, non-toxicity and non-inflammatory nature.

3.5.11. MoS2/reduced graphene oxide modified glassy carbon electrode A new matrix consisted of molybdenum disulfide-reduced graphene oxide hybrid (MoS2-rGO) was formed and characterized depicts a representative SEM image of the modified GCE electrode. A 3D spherelike architecture was observed with a complete overlapping of rGO and MoS2 nanosheets. FA molecules got oxidized on the surface of rGO & MoS2 modified GC electrode as shown in Supplementary Fig. 15. Modification of GCE with MoS2-rGO (MG) using drop casting allowed for the selective analysis of FA in the presence of a variety of interference species with detection limit 10 nM, linearity between 0.01 μM and 100 μM having sensitivity of 14 μA μM−1. Moreover, it successfully determined FA in human serum samples. It offered promising interfaces for bio-electrochemical applications [41]. Merits: Being two-dimensional, graphene provides a platform for 6

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pH 7.4 using scan rate of 50 mVs-1. FA got oxidized on the surface of SAOS modified carbon paste electrode as by cyclic voltammogram in Supplementary Fig. 17. Folic acid LOD was found to be 28.8 μM. The modified electrode (SAOSMCPE) exhibited good electrocatalytic activity for determination of FA. The same method can also be applied for sensing other analytes [45]. Merits: Easy to synthesise, showed wider linearity and lower detection limit. Demerits: Not applied for FA detection in different real samples, not studied interference due to other analytes except dopamine.

loading many different particles, and thus reveals the new pathways for the synthesis of functional nanocomposites with different catalytic, magnetic, and optoelectronic properties. Strong synergistic interaction between graphene and MoS2 can greatly enhance the catalytic activity and stability. Demerit: GCE has very slow electron transfer kinetics for most inner sphere processes (e.g. redox chemistry of many analytes and interferences), especially if it is clean and has very little oxygen functionality. Additionally, most species do not adsorb on it. 3.5.12. Electrochemical sensing of folic acid using Fe2O3 nanostructures GC electrode was modified with Fe2O3 nanofibers and its morphology was investigated by FE-SEM analysis. It revealed with an average fibre diameter of 288 nm with highly interconnections with each other. The current response was found linear in the FA range of 60–60000 nM. The modified electrode displayed an excellent sensitivity with detection limit of 60 nM. This modified electrode was applied for FA determination in human sera successfully even in the presence of 1000-fold excess of ascorbic acid [42]. Merits: It worked for broad linear range with no interference. Demerits: Fe2O3 nanofibres were physically adsorbed on the surface of GC electrode, thus led to lesser stability.

3.6.3. Calixarene chemically modified electrodes based Folic acid biosensor Voltammetric sensing of FA using plane CPE and electrode modified with calixarenes was compared. Two peaks for irreversible oxidation were observed. Out of the three calixarenes chosen for modification of the electrodes, p-tert-butyl-calix [6]arene modified electrode (CME-6) proved better for FA sensing. Chronocoulometric and DPV studies revealed that FA can assemble at CME-6 to form a monolayer whose electron transfer rate is 0.00273 s−1 with 2-electron/2-proton transfer for the peak at +0.71 V against SCE. DPV depicted the redox behaviour of folic acid on the surface of chemically modified carbon paste electrode (Supplementary Fig. 18). An adsorption equilibrium constant of 5 × 103 l/mol for maximum surface coverage of 2.89 × 10−10 mol/cm2 was obtained. Linearity in the lower range of concentration found to be 8.79 × 10−12 M to 1.93 × 10−9M having correlation coefficient of 0.9920 using adsorptive stripping voltammetry. Detection limit was found to be 1.24 × 10−12 M. It was applied for the determination of FA in different samples, viz. serum, asparagus, spinach, oranges and multivitamin preparations [46]. Merits: The key benefits of carbon paste electrodes include low cost, simple design, low background current, ability to introduce various modifiers during paste preparation, easy removal of electrode surface layer, low ohmic resistance and wide potential range. Demerits: CPE being prone to mechanical damage during use, so cannot be used in organic solvents.

3.5.13. Cu doped SnO2 nanoparticles modified glassy carbon electrode Cu doped SnO2NPs based folic acid biosensor was fabricated. Cu doped SnO2NPs were characterized by XRD & TEM studies which revealed their size in the range of 5−9 nm. SEM and EDX-mapping were carried out to investigate the nature of Cu dopant onto SnO2. From these measurements it was observed that the copper and tin were homogenously dispersed in the doped particles. TEM image of pure SnO2 samples (Fig. 2(B)) showed nearly spherical shaped nanoparticles with average particles size of 20 nm. The biosensor exhibited the detection limit of 0.02 nM with wide linear range of 1 × 10−10 to 6.7 × 10−5 M. Biosensor was applied for the determination of FA in pharmaceutical sample with satisfactory recovery in the presence of ascorbic acid [43]. Merits: It worked for a wider linear range having very low detection limit. Demerits: As Cu doped SnO2NPs were just casted on GC electrode, they may leach out, hence, does not provide much stability.

3.7. Boron doped diamond electrode based Folic acid sensor Cyclic, linear sweep and adsorptive stripping voltammetric studies were done to characterise electrochemical properties of FA in pH range 1.0–9.0. On the surface of boron doped diamond electrode (BDD) FA molecules got adsorped and oxidized as evidenced by graphs (Supplementary Fig. 19). In square-wave stripping mode, FA yielded well-defined voltammetric responses in both 0.1 M perchloric acid and 0.1 M Britton-Robinson buffer, pH 6.0 with limits of detection 0.035 μg/mL (7.93 × 10−8 M) and 0.14 μg/mL (3.2 × 10−7 M), respectively, after an accumulation of 120 s at open-circuit condition. Practical applicability of the newly developed approach was verified by the direct assays of tablet dosage forms [47]. Merits: Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. Demerits: BDD electrodes exhibit major drawbacks of mechanical and chemical stability.

3.6. Carbon Paste Electrode based Folic acid biosensor 3.6.1. Nickel ions dispersed poly(o-anisidine) film modified carbon paste electrode based FA biosensor On the surface of CPE, poly(o-anisidine) (POA) was formed by successive Cyclic voltammetry in monomer solution containing sodium dodecyl sulfate (SDS). Ni (II) ions were deposited on electrode by immersion of the polymeric modified electrode having amine group in 0.1 M Ni (II) ion solution. Ni(OH)2/NiOOH couple exhibited good redox behaviour. FA got oxidized by Ni(OH)2 in the POA film deposited on the surface of CPE as shown in Fig….. The catalytic oxidation peak current of FA was found linear dependent on its concentration and a linear calibration curve was obtained in the range of 0.1–5 mM with a correlation coefficient of 0.9994. The LOD was determined as 0.091 mM. This electrocatalytic oxidation was used as simple, selective and precise voltammetric method for determination of FA in pharmaceutical preparations [44]. Merits: The working electrode was prepared using electropolymerisation, hence showed more stability. Demerits: It was not applied for real sample analysis of folic acid.

3.8. Mercury film electrode based Folic acid sensor To determine FA even at the submicromolar concentration levels, stripping method was developed. It was based on controlled adsorptive accumulation of FA at the hanging mercury drop electrode followed by DPV measurement of the adsorbate. CV at pH 7.1(0.2 ML−1 KH2PO4/

3.6.2. Sodium alpha olefin sulphonate modified carbon paste electrode based sensor CPE was modified with sodium alpha olefin sulphonate (SAOS) for electrochemical sensing FA, in 0.2 M phosphate buffer solution (PBS) at 7

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NaOH buffer solution) showed four cathodic peaks at −0.57, −0.75, −1.18 and −1.39 V. The second peak, (at −0.75 mV), was the most useful for analytical purposes (more sensitive, quasi reversible and well defined) and it was used for quantifying FA by DPV adsorptive stripping voltammetry in four types of pharmaceutical multivitamin preparations. Sequential determination of FA with AA is also possible. The average content of FA found in these medicines by HPLC was 4.81 ± 0.09 mg and by voltammetry, 4.87 ± 0.09 mg. The current method is very simple, efficient and does not involve time-consuming separation steps [48]. Merits: It includes the ability of metals to dissolve in mercury—resulting in the formation of an amalgam—and the ability to easily renew the surface of the electrode by extruding a new drop. Demerits: Mercury film electrode exhibited limited anodic window due to mercury oxidation and show high toxicity.

Fig. 20a). Supplementary Fig. 20b represented the uniform MWCNTs film formed from a well dispersive MWCNTs solution. In addition, Supplementary Fig. 20c showed WCNTs-PBCB nanocomposite with clusters of PBCB modified onto MWCNTs/ITO electrode. Further, in Supplementary Fig. 20c the quantity of PBCB is higher compared to the quantity formed on the bare electrode in supplementary Fig. 20a, which could be a evidence of higher deposition of PBCB in presence of MWCNTs. The MWCNTs-PBCB composite film exhibited promising enhanced electrocatalytic activity towards the biochemical compound FA. The electrocatalytic response of FA at PBCB, MWCNTs and MWCNTs-PBCB composite films have been studied using CV and DPV. The composite film of MWCNTs-PBCB deposited on the surface of GCE, catalysed the electrochemical reduction of folic acid as depicted in Supplementary Fig. 20d. The sensitivity of MWCNTs-PBCB film towards FA (177 μA mM−1 cm−2) was higher than the values obtained for PBCB (0.75 μA mM−1 cm−2) and MWCNT films (148 μA mM−1 cm−2). Similarly, the LOD of FA at MWCNTs-PBCB film (76 μM) was lower than the other two films. Further, the DPV and selectivity studies revealed that the MWCNTs-PBCB film was efficient for FA determination in real sample [51]. Merits: ITO is one of the most widely used transparent conducting oxides because of its two main properties: its electrical conductivity and optical transparency, as well as the ease with which it can be deposited as a thin film. Demerits: ITO electrode exhibits expensive and time-consuming multi-stage process.

3.9. Sol-Gel carbon ceramic electrode based Folic acid biosensor Using CV, FA was determined in terms of the electrochemical behaviour of studied at an over oxidized polypyrrole modified carbon ceramic electrode. The modified electrode presented a linear response range for FA concentration from 7 to 55 μM, 0.1–25 μM, and from 0.49 to 7.8 μM with LOD of 1.8 × 10−6, 3.1 × 10−8, and 2.7 × 10−7 for CV, DPV, and amperometric techniques, respectively. The biosensor showed that the OPPy-modified electrode had very high catalytic ability for electro-oxidation of FA. The modified electrode exhibited excellent sensitivity and stability. The application of the modified electrode was demonstrated for determination of FA in biological and other real samples [49]. Merits: The carbon ceramic electrodes are rigid, porous, easily modified chemically and have a renewable external surface. The electrodes offer higher stability than carbon paste electrodes, and they are more amenable to chemical modification than monolithic and (organic) composite carbon electrodes. Demerits: Carbon ceramic electrode are highly affected by the preparation method, type of precursor and catalyst, which directly influence the conductive properties, mechanical and morphological characteristics of electrode, and these are reflected in their electrochemical response.

3.12. DNA modified-pencil graphite electrode (PGE) based Folic acid biosensor An electrochemical DNA biosensor was desinged as a screening device for the rapid analysis of FA using a PGE modified with salmon sperm ds-DNA. The optimum combinations for the reaction was pH 4.8, DNA concentration of 24 μg/mL, deposition time of 304 s, and deposition potential of 0.60 V, by which the adenine signal was recorded as 3.04 μA. FA could be measure in the range of 0.1–10.0 μmol/L with LOD of 1.06 × 10−8 μmol/L. The relative standard deviations for ten replicate differential pulse voltammetric measurements of 2.0 and 5.0 μmol/L FA were 4.6 % and 4.3 %, respectively. The biosensor was successfully used to measure FA in different real samples [52]. Merits: PGE have several advantages over other carbon-based or commercial metal electrodes, including widespread availability, very low cost, and ease of modification. Demerits: PGE themselves possess numerous disadvantages, including structural fragility and the necessity of a specialized holding device. Furthermore, because each lead can only be used for a few scans, PGE introduces error as the same electrode cannot be used for an entire experimental series.

3.10. Lead film electrode based folic acid sensor An adsorptive stripping voltammetric procedure for the determination of FA at an in situ plated lead film electrode was described. Formation of lead film on a GC substrate and accumulation of FA was performed simultaneously from an acetate buffer solution of pH 5.6 at the potential −0.88 V. The calibration graph for an accumulation time of 300 s was linear from 2 × 10−9 to 5 × 10−8 mol L−1. The LOD was 7 × 10−10 mol L−1 and the relative standard deviation for 2 × 10−8 mol L−1 of FA was 3.9 %. The biosensor was applied to FA determinations in pharmaceutical preparations [50]. Merits: The lead film electrode exhibits the distinct advantages over toxic (mercury) materials i.e sample preparation, good reproducibility and electrochemical surface renewal. Demerits: The lead film electrode shows fouling of electrodeposited nanomaterials and lack of reproducibility due to intermetallic compounds.

3.13. Immunoassay based on electrochemical magneto sensor for Folic acid detection The immunological reaction for FA detection was performed, for the first time on the magnetic bead as solid support by the covalent immobilization of a protein conjugate BSA-FA on tosyl-activated magnetic bead. Further competition for the specific antibody between FA in the food sample and FA immobilized on the magnetic bead was achieved, followed by the reaction with a secondary antibody conjugated with HRP (Anti IgG-HRP). Then, the modified magnetic beads were easily captured by a magneto sensor made of graphite-epoxy composite (mGEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with electrochemical detection using magneto sensors was evaluated in spiked-milk samples and compared with a novel magneto-ELISA based on optical detection [53]. Merits: The magneto sensor strategy offers great promise for rapid,

3.11. Poly(brilliant cresyl blue)-multiwall carbon nanotube composite film modified indium tin oxide (ITO) electrode based folic acid biosensor Electrochemically active composite film which contained multiwalled carbon nanotubes (MWCNTs) incorporated with poly(brilliant cresyl blue) (PBCB) has been prepared on GCE and ITO electrode by potentiodynamic method. The SEM image showed the appearance of clusters of PBCB on the surface of ITO electrode (Supplementary 8

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reproducibility of 4–10 % [56]. Merits: It is simple method and does not require skilled personnel. Demerits: It requires time consuming sample preparation.

simple, cost-effective and on-site analysis of biological and food samples. Demerits: The magnetosensor has some drawbacks of low sensitivity, high noise and high cost.

3.15.3. Folate binding protein based optical folic acid biosensor A ligand binding assay based approach was used in detection of Bovine milk-derived folate binding protein. The assay was performed by optical biosensor using a monoclonal antibody. Samples preparation was done using direct buffer dilution and heat treatment. Analysis conditions were optimized and non-specific binding considerations were evaluated. Single laboratory validation performance parameters were reported and the relative affinities for the major endogenous reduced folate in milk support the attributes of the optical biosensor assay utilising folate binding protein as compared with the antibody [57]. Merits: It is easy to perform. Demerits: It is not an economic method for determination of folic acid.

3.14. A folic acid-based functionalized surface for biosensor systems Intricacy of a biosensor with the biological system provides specificity towards biological analyte of interest. A cantilever sensor using folic acid as functional component has been documented to detect nasopharyngeal (KB) cancer cells. Here, folic acid is conjugated to a titanium coated sensor surface using silane linker. ELISA was used to verify biological activity of sensor and KB cell binding experiments to access overall performance of cantilever sensor. However, cantilever sensors need to be supplemented with other sensor technologies and bioinformatics tools in wake of biological complexities of pathogen diagnosis which can potentially bridge the lacunae between biomarker research and pathological inferences of disease [54].

3.15.4. Carbon nanohorns supported interwoven titanate nanotubes based photo electrochemical sensor for Folic acid detection Herein, a biosensing platform based on interwoven titanate nanotubes and carbon nanohorns conjugates was fabricated for FA detection. Specifically, titanate nanotubes having reticulation structure were used as optoelectronic element, which showed good photocatalytic activity and high electron kinetics. Hierarchical-structured carbon nanohorns used to provide electron-transport medium to capture and transmit electrons from excited titanate nanotubes to the biosensing surface owing to the high conductivity and large surface area. Cyclic voltammetry exhibited the electrochemical behaviour of folic acid using Fe (CN)63−/4− as redox probe (Supplementary Fig. 21). The biosensor depicted a wide linear response for FA was range from 1 × 10−10 to 5 × 10−5 M with a LOD of (2.5 ± 0.005) × 10−11 M [58]. Merits: Optical biosensors for FA detection offer great advantages over conventional analytical techniques because they enable the direct, real-time and label-free detection of many biological and chemical substances. Their advantages include high specificity, sensitivity, small size and cost-effectiveness. Demerits: The optical biosensors are, expensive, loss of activity when they are immobilized on a transducer, tend to lose activity after a relatively short period of time.

3.15. Optical biosensor for folic acid detection 3.15.1. BSA-modified gold nanoclusters based fluorescence biosensor for Folic acid detection Gold nanoclusters based optical FA biosensor has been designed. With analysis of the fluorescence and absorbance spectra of the gold nanoclusters in the presence and absence of FA, the mechanism of florescence quenching of BSA-AuNCs by FA was investigated. At the pH of 7.4, the fluorescence quenching was well suited to Stern–Volmer equation with a wide linear response in the concentration range of 120.0 ng/mL to 33.12 μg/mL and the LOD of 18.3 ng/mL. Furthermore, the sensor was applied in detection of FA in pharmaceuticals samples [55]. Merits: It is simple & easy to execute. It is cost-effective. Demerits: It is non-specific for determination of folic acid. 3.15.2. Immunoaffinity based optical biosensor for folic acid detection A new optical method was designed for the quantification of FA in fortified food is presented. The optical biosensor was applied to determine FA concentration levels in milk powder, infant formula and cereal samples. It was based on measuring the complex of antibody with analyte which is immobilized on the surface of sensor chip. Accuracy of the method (88–101 %) was determined with the monitoring of FA in the real samples. The precision study of ten participants at four different laboratories showed relative standard deviations of 2–8 % and

3.16. ZnO nanowires array based folic acid biosensor Graphene foam (GF) was synthesized by chemical vapor deposition

Fig. 5. Representation of process of Gr/ZnONWAs/GF and electrochemical redox reactions of FA and UA on the surface of Gr/ZnONWAs/GF [59]. 9

10

0.5 0.3 0.6 – 0.6 0.8 0.86 0.015 −0.71 0.75 0.65 0.77 −0.2

4 × 10−6 7.50 × 10−6 1.6 × 10−11 1 × 10−6 6 × 10−8 1 × 10−7 2.3 × 10−7 9.53 × 10−6 1 × 10−6 1 × 10−6 1.5 × 10−5 7.5 × 10−5 3.3 × 10−8 5 × 10−5 1 × 10−5 0.091

2 × 10−4–0.1 1 × 10−4–0.1 0.1–5

2-mercaptobenzothiazole self assembled Au electrode Polyoxometalate polypyrrole AuNPs modified Au electrode AuNPs modified Au electrode Reduced graphene oxide (rGO)/AuE Mesoporous carbon/ glassy carbon electrode (GCE) Phosphomolybdic-polypyrrole film modified GCE Thiadiazole modified GCE Bismuth nanowires/ GCE Single walled carbon nanotubes (SWCNT)/GCE SWCNT ionic liquid paste/GCE CuO nanoleaves/MWCNT/GCE Hydroxyapatite NPs/GCE Phosphomolybdic acid-polypyrrole/graphene nanocomposite/GCE Platinum NPs/MWCNT/GCE MoS2/rGO/GCE Poly (o-anisidine)/sodium dodecyl sulfate/carbon paste electrode (CPE) Sodium α-olefin sulfonate modified/CPE Calixarene based chemical modified GCE

Boron doped diamond electrode Mercury film electrode Polypyrrole modified sol-gel carbon ceramic electrode Lead film electrode DNA modified-pencil graphite electrode BSA-FA tosyl- activated magnetic bead BSA modified gold nanocluster

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 20. 21. 22.

28. 29. 30. 31. 32. 33. 34.

26. 27.

23. 24. 25. 0.1–0.35 8.79 × 10−9–1.93 × 10−6 2.3 × 10−4–4.5 × 10−2 2.2 × 10−3–1 × 10−2 0.007–0.055 2 × 10−6–5 × 10−5 1 × 10−5–1 × 10−3 2 × 10−5–1.3 × 10−4 2.7 × 10−4–0.075

0.8

4.53 × 10−6

1.58 × 10−5–3.5 × 10−4 8 × 10−6–1.0 × 10−3 1 × 10−5–1 × 10−3 1 × 10−4–1 × 10−2 1 × 10−9–1 × 10−7 5 × 10−7–1 × 10−4 1 × 10−5–1 × 10−4 1 × 10−4–0.8 1 × 10−5–15 × 10−5 1 × 10−5–0.1 2 × 10−6–4 × 10−3 1 × 10−5–9 × 10−4 1 × 10−4–0.35 1 × 10−6–2 × 10−4

4.

0.114 0.71 1.5 −0.2 0.7 −0.88 4.5 – 0.2

0.0288 1.24 × 10−9 7.93 × 10−5 1.4 × 10−5 1.8 × 10−3 7 × 10−7 7.5 × 10−6 1.3 × 10−5 4.1 × 10−5

1.2 0.7 0.6

−0.25 −0.2 1.2

1 × 10−5 5 × 10−7 4.32 × 10−6

2 × 10−4–1 × 10−3 5 × 10−7–2.5 × 10−5 1.7 × 10−6–7.5 × 10−6

Carbon fibre microelectrode Mercury meniscus modified silver amalgam electrode Molecular imprinted polymer based carbon composite electrode Sol-Gel coated pencil graphite electrode (PGE)

1. 2. 3.

Potential (V)

Detection limit (μM)

Linear Range (μM)

Type of electrode

S.NO.

Table 1 A comparison table of various electrochemical biosensors for determination of folic acid.

30 120 – 300 60 – –

– 120

– 4.3 2.7 – 2.09 3.9 3.2 – 4

– – –

60 – 304 60 10 – 3 5 300 360 5 5 –

120

– 15 180

Time (s)

1.9 6.3 –

6.3 Nil 4.5 4.3 2.66 – 1.21 2.5 3.41 2.26 – 3.4 1.5

3

2.5 1.2 1.4

Precision (%)

6.0 7.1 7.4 5.6 – 7.5 7.4

7.4 4.0

7.4 7.4 7.0

7.4 – 4.8 7.4 7.0 – 7.2 4.5 5.5 5.5 7.0 7.0 7.0

2.5

1.8 5 7.8

pH

0.99 0.99 0.99 0.99 0.98 0.99 0.99

0.99 –

0.99 – 0.99

0.99 0.99 – 0.98 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99

0.99

0.99 – 0.99

Correlation Coefficient (R)

Pharmaceuticals Food and blood serum samples Pharmaceuticals Pharmaceuticals Biological samples Pharmaceuticals Pharmaceuticals Biological and food samples Pharmaceuticals

Pharmaceuticals Biological fluids Blood serum samples

Pharmaceuticals Foods and pharmaceuticals Foods and pharmaceuticals Blood serum samples Blood serum samples Pharmaceuticals Blood serum samples Pharmaceuticals Pharmaceuticals Food samples Pharmaceuticals Pharmaceuticals Blood serum samples

Blood serum samples

Biological samples Nutritional supplements Blood serum samples

Application

[47] [48] [49] [50] [52] [53] [55]

[45] [46]

[40] [41] [44]

[27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39]

[26]

[23] [24] [25]

References

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with low cost. Therefore, the upcoming research in FA biosensors would focus on fully automatic lab on chip devices so that they can be easily used by the patients at home or his/her bedside at hospitals [63]. Labs on chip devices represents various advantageous features like rapid response, low sample intake and cost effectiveness [64]. Additionally, the enzyme nanoparticles based FA biosensor could be designed to make the fabrication process simpler and effective [65,66].

(CVD) using nickel foam as the template. Then, ZnO nanowire arrays (ZnO NWAs) were grown on the GF by hydrothermal synthesis. Finally, graphene (Gr) was deposited on the ZnO NWAs by CVD to obtain the hybrid of Gr/ZnO NWAs/GF (Fig. 5). SEM images of Gr/ZnO NWAs/GF exhibited the 3D macroporous structure of nickel foam with the average pore diameter of ∼ 300 μm (Supplementary Fig. 22). TEM images revealed the diameter and length of ZnO nanowires as ∼ 50 nm and 2 μm, respectively. Due to large specific surface area and outstanding electric conductivity, the hybrid can be used for the determination of FA by CV and DPV. The results showed that ZnO NWAs were uniformly and vertically grown on the GF and Gr is deposited on the ZnO NWAs. Cyclic voltammogram depicted the oxidation of FA on the surface of Gr/ZnONWAs/GF. The sensitivity and the measured LOD of the hybrid for FA in the range of 0–60 μM are 0.18 μA μM− 1 and 1 μM, respectively [59]. Merits: The nanowires hybrid can accurately detect FA in the presence of uric acid, and the hybrid also shows good reproducibility and stability. Demerits: The major disadvantage of ZnO nanowires are low yield, less uniformity and complex synthesis procedure. Table 1 summarised the comparison table of various electrochemical biosensors for determination of FA. This comparison showed that gold electrode based FA biosensor worked in linear range 1 × 10-9 to 1 × 10-3 μM which was better than PGE (1.58 × 10-4 to 1 × 10−2 μM), CPE (2 × 10-4 to 5 μM) and glassy carbon electrode (8.79 × 10-9 to 0.8 μM), whereas PGE exhibited better LOD (1.6 × 10–11 μM) as compared to GCE (1.24 × 10-9 μM) and CPE (4.32 × 10−6 μM). The GCE electrode showed better potential range (-0.71 to 1.2 V) rather than CPE (-0.25 to 1.2 V), PGE (0.6 to 0.8 V) and Au electrode (0.3–4.5 V). The GCE electrode based biosensor revealed rapid response (5−360 s) besides AuE (60 s), PGE (120−304 s) and CPE (180 s).

Declaration of Competing Interest None. Acknowledgement This work was supported by a grant from University Grants Commission (UGC), New Delhi-110012. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2020.01.025. References [1] Y. Amitai, G. Koren, The folic acid rescue strategy, JAMA Pediatr. 169 (2015) 1083, https://doi.org/10.1001/jamapediatrics.2015.2235. [2] D. Taruscio, P. Carbone, O. Granata, F. Baldi, A. Mantovani, Folic acid and primary prevention of birth defects, BioFactors. 37 (2011) 280–284, https://doi.org/10. 1002/biof.175. [3] W.S. Beck, Vitamin B12 and Folic Acid, Blood, (1974), pp. 461–494, https://doi. org/10.1016/b978-0-12-595705-2.50020-3. [4] A.E. Czeizel, Folic Acid/Folic acid-containing multivitamins and primary prevention of birth defects and preterm birth, Prev. Nutr. Food Sci. (2009) 643–672, https://doi.org/10.1007/978-1-60327-542-2_25. [5] M. Lucock, Folic acid: nutritional biochemistry, molecular biology, and role in disease processes, Mol. Genet. Metab. 71 (2000) 121–138, https://doi.org/10. 1006/mgme.2000.3027. [6] E.H. Reynolds, B.B. Gallagher, R.H. Mattson, M. Bowers, A.L. Johnson, Relationship between serum and cerebrospinal fluid folate, Nature. 240 (1972) 155–157, https://doi.org/10.1038/240155a0. [7] L. Haandel, M.L. Becker, T.D. Williams, J.F. Stobaugh, J.S. Leeder, Comprehensive quantitative measurement of folate polyglutamates in human erythrocytes by ion pairing ultra-performance liquid chromatography/tandem mass spectrometry, Rapid Commun. Mass Spectrom. 26 (2012) 1617–1630, https://doi.org/10.1002/ rcm.6268. [8] A. Hoffbrand, D. Provan, Megaloblastic anaemia and miscellaneous deficiency anaemias, Oxford Medicine Online. (2014), https://doi.org/10.1093/med/ 9780199204854.003.220506_update_002. [9] F.M. Asrar, D.L. Oconnor, Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets, J. Nutr. Biochem. 16 (2005) 587–593, https://doi.org/10.1016/j.jnutbio.2005.02.006. [10] G.M. Brown, Biogenesis and metabolism of folic acid, Metabolic Pathways (1970) 383–410, https://doi.org/10.1016/b978-0-12-299254-4.50012-1. [11] R. Obeid, S.H. Kirsch, M. Kasoha, R. Eckert, W. Herrmann, Concentrations of unmetabolized folic acid and primary folate forms in plasma after folic acid treatment in older adults, Metabolism (2010), https://doi.org/10.1016/j.metabol.2010.06. 020. [12] V. Herbert, Metabolism of folic acid in man, J. Infect. Dis. 128 (1973), https://doi. org/10.1093/infdis/128.supplement_3.s601. [13] A. Hurdle, Folic acid (3HFA) absorption and jejunal biopsy in mild nutritional folic acid deficiency, Pathology. 2 (1970) 193–198, https://doi.org/10.3109/ 00313027009081207. [14] J. Mcguire, Folic Acid, Folates, and Cancer, Nutrition and Cancer Prevention Nutrition and Disease Prevention, (2005), https://doi.org/10.1201/ 9781420026399.ch8. [15] R. Matias, P.R.S. Ribeiro, M.C. Sarraguça, J.A. Lopes, A UV spectrophotometric method for the determination of folic acid in pharmaceutical tablets and dissolution tests, Anal. Methods 6 (2014) 3065, https://doi.org/10.1039/c3ay41874j. [16] K. Hoppner, B. Lampi, The determination of folic acid (Pteroylmonoglutamic acid) in fortified products by reversed phase high pressure liquid chromatography, J. Liq. Chromatogr. 5 (1982) 953–966, https://doi.org/10.1080/01483918208060626. [17] G. Kanjilal, S.N. Mahajan, G.R. Rao, Colorimetric determination of folic acid in pharmaceutical preparations, Analyst 100 (1975) 19, https://doi.org/10.1039/ an9750000019. [18] Z. Song, L. Wang, Chemiluminescence inhibition assay for folic acid using flow injection analysis, Phytochem. Anal. 14 (2003) 216–220, https://doi.org/10.1002/ pca.704. [19] U. Hla-Pe, Aung-Than-Batu, A fluorometric method for urinary folic acid

4. Conclusion By concluding out the above mentioned FA biosensors, It can be said that biosensing methods are much superior to traditional colorimetric, titrimetric, spectrophotometric, chromatographic methods and fluorescence methods for monitoring of FA. FA biosensors are found operate ideally within 3–300 s, in pH range 1.8–7.8, FA concentration range 8.71 × 10-9-5 mM, LODs between 1.6 × 10–11 to 0.091 mM and with working potential -0.88 to 4.5 V. Mercury meniscus based FA electrodes have shown linearity in the range 5 × 10-7 to 1 × 10-2 μM and LOD in the range 5 × 10-7 to 1 × 10–7μM. Carbon fibre microelectrode based FA biosensor has evidenced linearity 2 × 10-4 to 1 × 10-3μM and LOD 1 × 10-3μM. Besides, PGE based electrodes have shown linear range 1.58 × 10-5 to 1 × 10-2 μM having LOD 4.53 × 10-6 to 1.6 × 10–11 μM. However, gold electrode-based FA sensors have shown linearity 1 × 10-9 to 1 × 10-3 μM and detection limit 7.5 × 10-6 to 1 × 10-6 μM. Carbon paste electrode based FA sensors has displayed linear range 0.1–5 μM and LOD 0.09 to 0.02 μM. Glassy carbon electrode based FA sensors has shown linear range 8.79 × 10-6 to 0.1 μM having LOD 1.24 × 10-9 to 10-5 μM. To summarize the analytical performance of different types of electrodes we have evidenced the wider linear range from Au electrodes (1 × 10-9 to 1 × 10-3 μM) and low detection limit from PGE (1.6 × 10−11μM). 5. Future perspective The FA biosensors are considered as an important device in clinical analysis of a large number of diseases such as cardiovascular diseases, neural tubes disorder, macrocystic anaemia, depression and pregnancy complications. Therefore there is a need to develop most effective and precise device for effective monitoring of the FA. The already available devices are not portable and also fail to real time monitoring of FA. Moreover, they can’t be used at home by patients. To keep in mind these drawbacks, there is a need to develop miniaturised FA biosensor 11

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