Recent Advances in Enhancement Strategies for Electrochemical ELISA-Based Immunoassays for Cancer Biomarker Detection

Electrochemical enzyme-linked immunosorbent assay (ELISA)-based immunoassays for cancer biomarker detection have recently attracted much interest owing to their higher sensitivity, amplification of signal, ease of handling, potential for automation and combination with miniaturized analytical systems, low cost and comparative simplicity for mass production. Their developments have considerably improved the sensitivity required for detection of low concentrations of cancer biomarkers present in bodily fluids in the early stages of the disease. Recently, various attempts have been made in their development and several methods and processes have been described for their development, amplification strategies and testing. The present review mainly focuses on the development of ELISA-based electrochemical immunosensors that may be utilized for cancer diagnosis, prognosis and therapy monitoring. Various fabrication methods and signal enhancement strategies utilized during the last few years for the development of ELISA-based electrochemical immunosensors are described.


Introduction
Cancer is one of the major causes of mortality in the world. Many factors, including exposure to cancer-causing reagents, exposure to radiation, infections, genetic modifications, etc., can disrupt the cells and result in their modification and proliferation causing the generation of cancer in different parts of the body. Its diagnosis based on visual symptoms is not recommended as such symptoms appear in later stages of cancer, when there are no efficient therapies. Thus, it is advised to diagnose it in early stages, when useful treatment is possible, in order to achieve longer survival of cancer patients [1]. To achieve early stage diagnosis researchers have proposed the use of proteins and oligonucleotides released in the body during the early stages of cancer and not present in the same concentrations in healthy individuals. Such molecules are known as biomarkers and different types of cancers release different biomarkers, whose detection and estimation can provide very valuable information regarding cancer type and its stage. Thus, it is very important to develop systems, which are simple, low cost and can provide sensitive and specific estimation of such biomarkers [2]. Further, taking into account population and cancer stage variability as well as low levels of biomarkers in early stages in cancer, it is recommended to identify and test panels of multiple biomarkers for better accuracy in diagnosis. Also, it is desired to detect these biomarkers in a non-invasive or minimally invasive manner with high selectivity, sensitively and free from false positives and false negatives. Commonly employed methods of cancer detection such as enzyme-linked immunosorbent assay (ELISA), western blotting, at faster rate of analysis along with ease of fabrication, measurement and mass production at low cost [9][10][11][12]. Keeping these advantages in mind, researchers have recently focused on the development of electrochemical ELISA-based systems to combine the advantages of sandwich assays used in optical ELISA and electrochemical detection [13]. Electrochemical ELISA enjoys the specificity and signal amplification obtained by the use of synchronized binding of the recognition molecule and detection molecule, along with high sensitivity, low detection limit, easy handling and easy detection in miniaturized format provided by electrochemical detection. With evolving material and surface chemistries along with advancing bio-and nano-technologies, electrochemical ELISA based immunosensors have been gaining much interest and promising to replace traditionally used optical ELISA to achieve faster, more sensitive, cheaper and reliable detection of cancer biomarkers in for early stage diagnosis [14,15]. The present review describes various new ways reported by researchers in the last 3 to 4 years for developing and improving sandwich-based electrochemical ELISA for cancer biomarker detection. Authors in many of these reports validated their approaches in spiked/real samples in vitro. There is no information regarding commercialization of any of these sensors at present, however these reports may pave way for better and faster diagnostic of cancer at earlier stages in the near future. Also, there are various useful reviews that have been published for electrochemical immunosensor-based cancer biomarker detection in past using nanoelectrodes, arrays and microfluidics [16][17][18]. Thus, in the future, combining the new advancements in sensor surfaces and detection probes described here with nanoelectrode arrays or microfluidics will further enhance the chances of achieving better sensitivity and detection limits required for early stage measurements of biomarkers.

Electrochemical Sandwich ELISA
Electrochemical sandwich ELISA is a branch of electrochemical immunoassays where the recognition of a desired target is done using a traditional sandwich assay and detection is achieved using an electrochemical method [19][20][21]. These immunoassays mainly involve three layers: immobilized biorecognition molecule (probe), target analyte that binds specifically to the biorecognition molecule, followed by binding of a secondary recognition molecule with an electrochemically active signal tag. For signal measuring, the electrochemical signal tag either provides the signal directly or a reaction with a substrate is induced afterwards [22,23]. The generated signal is directly proportional to the analyte concentration. This type of sensing involving sandwiching of target analyte between two highly specific capturing and recognition molecules, provides a high level of sensitivity and specificity and makes it suitable for early stage detection of cancer biomarkers [14]. For capturing and recognition molecules one can utilize combinations of suitable molecules ranging from antibodies, aptamers, DNA base sequences, bacteriophages, peptide nucleic acid sequences, etc. [2]. And for sensitive detection researchers have utilized various tags involving redox enzymes, metallic particles, quantum dots, etc., which they have used directly or in combination with another matrix for enhanced loading [6,14,24,25]. Other than these, the activity and recognition ability of developed sensors also depend on how and where the capturing molecule is immobilized and how well it can interact with the target analyte. Thus, innovative methods for binding of capturing agents on the desired surface (hereafter referred to as matrix, to reflect the modification of the electrode with different molecular and polymeric layers) for sensitive analyte capture are required for developing novel immunosensors. Further, the selection and development of a suitable matrix for binding of capturing agent is crucial to achieve optimum response from the assay [5]. Thus, for development of novel electrochemical ELISA-based immunosensors, research groups are working on finding newer and better matrices along with novel methods of binding capturing agents on the desired matrices and have proposed numerous approaches for higher binding of capturing molecules with better retained activity. Researchers have also proposed new and innovative methodologies to amplify the signal generated by binding events of target and recognition molecule. This review will focus mainly on the recent advances made by various groups in methods of making such electrochemical ELISA-based immunosensors for cancer biomarkers detection using innovative surface chemistries and materials along with their measuring methodology and response to analyte. For development of enhancement strategies, researchers have utilized various biomarkers such as CEA, AFP, PSA HER2, SCC, CA 125, CA 19-9, etc. Among these biomarkers CEA, AFP and PSA have gained much attention as model biomarkers for developing enhancement strategies. In a normal person, the cut-off concentration for CEA, AFP, CA 125 and PSA are found to be 3 ng/mL, 10 ng/mL, 46 U/mL and 4 ng/mL, respectively, and higher concentrations are oftern related to cancer stages. Immunosensors and enhancement strategies are normally investigated keeping these ranges in mind; the immunosensor is useful if it can detect concentrations lower than the cut off limit.

General Mechanism of Enhancement Strategies
In sandwich-based electrochemical ELISA, signals can be enhanced by increasing the capturing efficiency via the use of better antibodies or by their higher loading on sensor surface. Signals can also be enhanced via detection probes containing a larger number of detection tags. Thus, sensor surfaces where capture antibodies are immobilized and detection probes containing detection antibodies and tags play the main roles in achieving enhancement in signal. In general, enhancement in response has been achieved using modified sensor surfaces or improved detection probes. Using modified surfaces, researchers have tried to increase the surface area via use of nanomaterials and their composites, thus resulting in higher loading of antibodies. Also attempts were made to immobilize capturing antibodies in the desired orientation for enhance capturing efficiency, thus resulting in signal enhancement. In use of improved detection probes, researchers have utilized high surface area of nanomaterials and composites for loading of larger number of detection antibodies with tags or catalytic materials. On interaction of detection probe with antibody-antigen complex, one of the detection probe binds to antigen but many more are also available for further catalytic reaction of the detection substrate. In case of enzymatic tags, a larger number of tags results in larger conversion of specific analyte, thus resulting in larger quantity of detection molecules. Also, in the case of nanomaterial based catalytic tags on detection probe, large number of tags result in larger catalytic conversion of target substrate, thus resulting in higher response. Figure 1 shows the general detection strategy for optical and electrochemical ELISA. methodology and response to analyte. For development of enhancement strategies, researchers have utilized various biomarkers such as CEA, AFP, PSA HER2, SCC, CA 125, CA 19-9, etc. Among these biomarkers CEA, AFP and PSA have gained much attention as model biomarkers for developing enhancement strategies. In a normal person, the cut-off concentration for CEA, AFP, CA 125 and PSA are found to be 3 ng/mL, 10 ng/mL, 46 U/mL and 4 ng/mL, respectively, and higher concentrations are oftern related to cancer stages. Immunosensors and enhancement strategies are normally investigated keeping these ranges in mind; the immunosensor is useful if it can detect concentrations lower than the cut off limit.

General Mechanism of Enhancement Strategies
In sandwich-based electrochemical ELISA, signals can be enhanced by increasing the capturing efficiency via the use of better antibodies or by their higher loading on sensor surface. Signals can also be enhanced via detection probes containing a larger number of detection tags. Thus, sensor surfaces where capture antibodies are immobilized and detection probes containing detection antibodies and tags play the main roles in achieving enhancement in signal. In general, enhancement in response has been achieved using modified sensor surfaces or improved detection probes. Using modified surfaces, researchers have tried to increase the surface area via use of nanomaterials and their composites, thus resulting in higher loading of antibodies. Also attempts were made to immobilize capturing antibodies in the desired orientation for enhance capturing efficiency, thus resulting in signal enhancement. In use of improved detection probes, researchers have utilized high surface area of nanomaterials and composites for loading of larger number of detection antibodies with tags or catalytic materials. On interaction of detection probe with antibody-antigen complex, one of the detection probe binds to antigen but many more are also available for further catalytic reaction of the detection substrate. In case of enzymatic tags, a larger number of tags results in larger conversion of specific analyte, thus resulting in larger quantity of detection molecules. Also, in the case of nanomaterial based catalytic tags on detection probe, large number of tags result in larger catalytic conversion of target substrate, thus resulting in higher response. Figure 1 shows the general detection strategy for optical and electrochemical ELISA.

Matrix Selection, Modification and Development of Immunosensors
The development and properly working immunosensors involves the selection and preparation of a binding matrix followed by immobilization of capturing molecule on the surface of the electrode. The matrix for an electrochemical sensor can comprise monolayers, polymers, carbon based materials, nanomaterials or their composites [13,14,25,26]. Most commonly utilized capturing molecules include antibodies, antibody fragments, DNA/RNA aptamers and peptide aptamers, which are immobilized directly on a conducting/semiconducting electrode surface or on a pre-modified electrode surface via physical, entrapment or covalent methods [27]. Other than these,

Matrix Selection, Modification and Development of Immunosensors
The development and properly working immunosensors involves the selection and preparation of a binding matrix followed by immobilization of capturing molecule on the surface of the electrode. The matrix for an electrochemical sensor can comprise monolayers, polymers, carbon based materials, nanomaterials or their composites [13,14,25,26]. Most commonly utilized capturing molecules include antibodies, antibody fragments, DNA/RNA aptamers and peptide aptamers, which are immobilized directly on a conducting/semiconducting electrode surface or on a pre-modified electrode surface via physical, entrapment or covalent methods [27]. Other than these, researchers have also employed oriented biomolecular immobilization approaches either by using engineered capturing molecules, or by using molecules such as protein A, which allows the binding of antibodies in an 'upright' position for best activity. In any type of chosen method for binding of capturing molecule, the main emphasis during immobilization is to retain or enhance the capturing molecule activity and stability. Further, binding can also be characterized based on the chosen matrix [27].
In one example, Gao et al. utilized graphite to prepare GO, which was then reduced and nitrogen doped before coating on a glassy carbon electrode (GCE) for antibody binding via glutaraldehyde chemistry [28]. They further showed the use of β-cyclodextrin-graphene (β-CD-GR) for GCE coating and binding of capture antibody (Ab1)-adamantine (ADA) via physical adsorption [37]. A few other researchers have also utilized the CD-GS based matrix for immunosensor development [38,39]. Li et al. described another approach where single walled carbon nanotubes (SWCNTs) were mixed with L-cysteine modified chitosan (CS) to obtained thiol terminated CSSH-SWCNTs, which can be immobilized on gold surfaces from one side and can be employed for gold nanoparticles (AuNPs) binding on other side for larger surface area and Ab1 binding [40]. Similar to this, Wang et al. reported the use of AuNPs decorated mercapto-functionalized graphene sheets (Au@SH-GS) as matrix on GCE for Ab1 binding [41].
Other than these nanomaterials and composites, other materials such as polymers, magnetic materials, electrodeposited films and monolayers has also been utilized for immunosensor development. Wang et al. described the use of Ab1 tagged Dynabeads conjugates [59], while Zhou et al. described CD coated GCE electrode for immunosensor development [60]. Further, PAMAM modified GCE [61], cysteine monolayer on gold [62], etc. have also been utilized for advanced immunosensor development. Table 1 summarizes the various approaches used for immunosensor development. Illustration of the stepwise process for PSA immunosensor fabrication. Briefly, to prepare sensor electrode, physical mixture of MWCNTs and IL was coated onto GCE which was then modified with PAMAM decorated gold nanoparticles via phthaloyl chloride chemistry. Phthaloyl chloride chemistry was further utilized for thionine and anti-PSA immobilization. Reproduced with permission from [42].

Figure 2.
Illustration of the stepwise process for PSA immunosensor fabrication. Briefly, to prepare sensor electrode, physical mixture of MWCNTs and IL was coated onto GCE which was then modified with PAMAM decorated gold nanoparticles via phthaloyl chloride chemistry. Phthaloyl chloride chemistry was further utilized for thionine and anti-PSA immobilization. Reproduced with permission from [42].   Au@N-GQD HAuCl 4 was added to N-GQD, pH adjusted to 10 using NaOH followed by autoclaving at 160

Electrochemical ELISA Based Detection
For signal detection in electrochemical ELISA-based sensors, researchers have explored the use of various electrochemical amperometric and voltammetric techniques including differential pulse voltammetry (DPV), linear sweep voltammetry (LSV), stripping voltammetry and square-wave voltammetry (SWV). In general, once the analyte is captured, the sensor is incubated with the detection molecule tagged with an electroactive agent, such as a redox molecule, nanoparticle, and quantum dot, etc., or with an enzyme capable of generating electroactive species for signal measurement [17]. Use of redox enzyme-based indicator systems is most common and wildly applied in electrochemical ELISA-based immunosensors [105]. Traditionally a 1:1 ratio of redox enzyme and detection molecule is used for amplification and signal measurement. With the advances in material science and chemistry newer nano and hybrid materials have been explored in recent years to enhance the amplification of the signal. These advanced materials act as carriers for loading of multiple enzyme molecules and thus enhance the signal [10,24,25]. However, the use of redox tags and enzyme-mimicking molecules are also receiving much attention in the development of advanced immunosensors. The use of nano labels have also provided the opportunity to achieve better signals and to develop better immunosensors [106]. The signal is usually measured via an amperometric or voltammetric technique using a potentiostat or other low cost electrochemical systems, mainly in a three-electrode configuration. The following sections have been divided in a way to provide more details of different strategies employed for enhanced signal detection. Table 2 shows the details of various strategies used for the development of detection probes for enhanced detection. Furthermore, Table 3 shows the various characteristics of the developed immunoassays using electrochemical ELISA.   Anti-CA15-3/NP-PtFe NP-PtFe By removing Al using NaOH from ternary PtFeAl alloy with 80%Al [31] Anti-AFP/PdNi/N-GNRs PdNi/N-GNRs    PdCu-anti-CEA PdCu Using AA as reducing agent and HDPC as growth inhibitor [95] anti-AFP-GNPs-HRP GNP Citrate reduction [96] anti-AFP/HRP-Au@ZnO Au@ZnO C 18

Redox Enzyme Based Detection
The majority of immunosensors till date use redox enzymes for signal amplification and to enhance sensitivity of the immunoassay [22]. In such assays, the detection molecule, which binds to the antigen at a second binding site, is either tagged directly to a redox enzyme or is labeled with a tag capable of binding a modified redox enzyme. After redox enzyme binding, the enzyme catalyzes its substrate and generate an electroactive product, whose measurement give the information regarding the target analyte. Redox enzymes are used either as free molecules or after loading them on metallic or carbon-based nanomaterials for higher signal.

Free Redox Enzyme and Redox Enzyme with Nanomaterial Based Enhancement
In a free redox enzyme and redox enzyme with nanomaterial approach, redox enzyme tagged with detection antibody catalyzes its substrate to generate the electrochemical response. This section describes the various approaches investigated by researchers for loading of redox enzymes onto various nanomaterials for enhancing their concentration during the immunoassay, which in turn increases the signal response. Among various nanomaterials, gold nanoparticles have been investigated most. They have also been used in combination with CNTs and other composites. In the last few years, the use of free enzymes for enhancement strategy is rarely utilized as more advanced strategies have been developed. In one such study of free enzyme based system, Patris et al. utilized the horseradish peroxidase (HRP) tagged detection antibody (Ab2) for signal detection in HER2 immunosensor. For signal enhancement, the antibody-antigen complex was incubated with detection probe for 20 min followed by testing in citrate buffer containing 2.5 mM hydrogen peroxide (H 2 O 2 ) and the reduction current for added hydroquinone (HQ) was monitored at −280 mV. Results showed detection of HER2 at two concentrations: 1 and 200 µg/mL [63]. For better enhancement, researchers have developed many nanomaterial-tagged redox enzymes based strategies and achieved improved detection limit and sensitivity. In one such study, Feng et al. utilized physically loaded Ab2 and HRP onto AuNPs-PANI@CNTs nanocomposites for signal enhancement in their immunosensor for CEA detection [47]. Detection probe was developed by chemical reduction of aniline in CNT presence followed by electrostatic assembly of AuNPs. During immunoassay, Ab1-antigen complex is incubated with detection probe for 55 min at 37 • C and CEA detection down to 0.008 ng/mL via DPV was achieved in phosphate buffer saline (PBS) containing 4 mM H 2 O 2 . They observed two linear ranges, and ascribed the lower concentration range to isadsorption-controlled processes on the electrode, whereas linearity in higher concentrations was attributed to diffusion controlled processes on the electrode. In other study, Kayosi et al. described the use of HRP-prostate specific antigen (PSA) aptamer-modified AuNP-PAMAM conjugate for signal enhancement [70]. They utilized glutaraldehyde chemistry for immobilizing PSA aptamer and HRP-PSA aptamer (prepared using streptavidin-biotin coupling) onto detection probe. With this probe they achieved PSA detection down to 10 fg/mL when tested by DPV scanning. Figure 3 shows the schematic for detection probe development. Further, AuNPs have been utilized by Zhang et al. in the development of AuNPs modified SBA-15 (Au@SBA-15) based detection probe for carbohydrate antigen 19-9 (CA 19-9) estimation [74]. Ab2-HRP was conjugated onto Au@SBA-15 via Au-NH 3+ or Au-SH affinity and enhanced direct electron transfer (DET) was utilized for signal enhancement. With such probe they were able to achieve CA 19-9 detection down to 0.01 U/mL. The use of HRP modified hollow AuNPs has been described by Li et al. for the development of an AFP immunosensor. In their method, hollow AuNPs were synthesized by HAuCl4 reduction in N2 environment using sodium borohydride (NaBH4), sodium citrate and CoCl2·6H2O mixed solution and then modified with L-cysteine modified HRP-NPs. Physically immobilized Thi-anti-AFP based probe then achieved AFP detection down to 8.3 pg/mL when incubated with Ab1-antigen for 30 min at room temperature and the DPV signal was recorded in the presence of H2O2 [40]. Figure 4 shows the schematic for HRP-HRP-NPs-hollow AuNPs-Thi@anti-AFP bioconjugates development. Moving away from gold, Wang et al. described the use of Fe3O4 and HRP modified mesoporous silica nanoparticles (MSNs) based detection probe for an AFP immunosensor. The detection probe with Ab2 and HRP were immobilized using glutaraldehyde chemistry, and showed AFP detection down to 4 pg/mL when tested via CV in PBS with and without 5 mmol/L H2O2 [64]. In another study, Wang et al. described silver nanoparticles (AgNPs) based detection probe for CEA estimation. For probe development anti-CEA and glucose oxidase (GOD) were physically immobilized onto Ag nanospheres prepared via an ethylene glycol (EG) and poly(vinyl pyrrolidone) (PVP) assisted method. For immunoassay, probe was incubated with Ab1-antigen for 1 h at 4 °C and the resulting complex was tested via DPV. With such probe they achieved CEA detection down to 0.27 pg/mL [56]. Further, Zhou et al. utilized HRP tagged BSA-nanosilver microspheres (Ag@BSA) to quantify CEA. Using enzymatic precipitation and amplification of tyramine signal, they achieved CEA detection down to 5.0 pg/mL via DPV method [108]. Tang et al. utilized HRP and Ab2 modified magnetic The use of HRP modified hollow AuNPs has been described by Li et al. for the development of an AFP immunosensor. In their method, hollow AuNPs were synthesized by HAuCl 4 reduction in N 2 environment using sodium borohydride (NaBH 4 ), sodium citrate and CoCl 2 ·6H 2 O mixed solution and then modified with L-cysteine modified HRP-NPs. Physically immobilized Thi-anti-AFP based probe then achieved AFP detection down to 8.3 pg/mL when incubated with Ab1-antigen for 30 min at room temperature and the DPV signal was recorded in the presence of H 2 O 2 [40]. Figure 4 shows the schematic for HRP-HRP-NPs-hollow AuNPs-Thi@anti-AFP bioconjugates development. Moving away from gold, Wang et al. described the use of Fe 3 O 4 and HRP modified mesoporous silica nanoparticles (MSNs) based detection probe for an AFP immunosensor. The detection probe with Ab2 and HRP were immobilized using glutaraldehyde chemistry, and showed AFP detection down to 4 pg/mL when tested via CV in PBS with and without 5 mmol/L H 2 O 2 [64]. In another study, Wang et al. described silver nanoparticles (AgNPs) based detection probe for CEA estimation. For probe development anti-CEA and glucose oxidase (GOD) were physically immobilized onto Ag nanospheres prepared via an ethylene glycol (EG) and poly(vinyl pyrrolidone) (PVP) assisted method. For immunoassay, probe was incubated with Ab1-antigen for 1 h at 4 • C and the resulting complex was tested via DPV. With such probe they achieved CEA detection down to 0.27 pg/mL [56]. Further, Zhou et al. utilized HRP tagged BSA-nanosilver microspheres (Ag@BSA) to quantify CEA. Using enzymatic precipitation and amplification of tyramine signal, they achieved CEA detection down to 5.0 pg/mL via DPV method [108]. Tang et al. utilized HRP and Ab2 modified magnetic nanoparticles (MNPs) based strategy for PSA, PSMA, IL-6, and PF-4 estimation in ab array format. HRP and Ab2 were tagged onto MNP via biotin-streptavidin chemistry. Using HRP tag and added HQ as mediator they detected the target via DPV in the presence of H 2 O 2 and achieved detection down to pg/mL range for all four analytes [81]. In other study, Uludag et al. utilized HRP and anti-PSA tagged AuNPs as detection probe TMB as mediator to achieve detection down to 0.2 ng/mL when tested via amperometric at −0.1 V in the presence of H 2 O 2 [82].

Redox Enzyme with Carbon Material Based Enhancement
To enhance the response of immunosensors in electrochemical ELISA, various carbon-based nanomaterials have been explored to increase the loading of redox enzyme tagged detection antibody probes. This section describes various carbon-based material investigated by researchers to enhance the sensitivity of immunosensor. Among various carbon-based nanomaterial, graphene oxide and reduced graphene oxide have gained maximum attention in recent years. Other than graphene, various carbon-based materials such as CNTs, MWCNTS, nanodots and nanocomposites, etc. have also been utilized for developing detection probes to achieve high sensitivity. Huang et al. described a Ag/Au NPs-graphene based enhancement strategy for CEA immunosensor: a 1,5-diaminonaphthalene (DN)-based Ag/Au-DN-GR probe was prepared simply by mixing Ag/Au (prepared via reduction) with DN-GR. The immunoassay with physically adsorbed anti-CEA onto Ag/Au-DN-GR showed CEA detection down to 8 pg/mL when incubated with Ab1-antigen for 40 min [34]. Similarly, a Au@Pd-GR composite was used by Yang et al. for detection probe development by immobilizing Thi, HRP and anti-CA19-9. It was observed that synergy between Au@Pd-GR and HRP resulted in three times higher response in the presence of H2O2 and the sensor exhibited CA19-9 detection down to 0.006 U/mL [72].
GR-PAMAM dendrimer conjugate based detection probe development was described by Shen et al. GR and PAMAM were conjugated via EDC/NHS chemistry and then utilized for anti-AFP and HRP binding using glutaraldehyde cross-linking. With this simple probe and hydroquinone as detection molecule, they achieved amperometric detection of AFP down to 0.45 ng/mL [50]. In other study, Yang et al. described the development of duel enzyme bio-catalyzed precipitation of 4-CN based immunosensor for α-fetoprotein (AFP) detection. For probe, HRP, GOD and anti-AFP were

Redox Enzyme with Carbon Material Based Enhancement
To enhance the response of immunosensors in electrochemical ELISA, various carbon-based nanomaterials have been explored to increase the loading of redox enzyme tagged detection antibody probes. This section describes various carbon-based material investigated by researchers to enhance the sensitivity of immunosensor. Among various carbon-based nanomaterial, graphene oxide and reduced graphene oxide have gained maximum attention in recent years. Other than graphene, various carbon-based materials such as CNTs, MWCNTS, nanodots and nanocomposites, etc. have also been utilized for developing detection probes to achieve high sensitivity. Huang et al. described a Ag/Au NPs-graphene based enhancement strategy for CEA immunosensor: a 1,5-diaminonaphthalene (DN)-based Ag/Au-DN-GR probe was prepared simply by mixing Ag/Au (prepared via reduction) with DN-GR. The immunoassay with physically adsorbed anti-CEA onto Ag/Au-DN-GR showed CEA detection down to 8 pg/mL when incubated with Ab1-antigen for 40 min [34]. Similarly, a Au@Pd-GR composite was used by Yang et al. for detection probe development by immobilizing Thi, HRP and anti-CA19-9. It was observed that synergy between Au@Pd-GR and HRP resulted in three times higher response in the presence of H 2 O 2 and the sensor exhibited CA19-9 detection down to 0.006 U/mL [72].
GR-PAMAM dendrimer conjugate based detection probe development was described by Shen et al. GR and PAMAM were conjugated via EDC/NHS chemistry and then utilized for anti-AFP and HRP binding using glutaraldehyde cross-linking. With this simple probe and hydroquinone as detection molecule, they achieved amperometric detection of AFP down to 0.45 ng/mL [50]. In other study, Yang et al. described the development of duel enzyme bio-catalyzed precipitation of 4-CN based immunosensor for α-fetoprotein (AFP) detection. For probe, HRP, GOD and anti-AFP were immobilized via EDC/NHS chemistry on carboxylated SWCNHs. With their probe they achieved AFP detection down to 0.33 pg/mL [73].

Redox Marker Based Detection
Other than redox enzymes, researchers have employed the use of redox active tags such as nanoparticles, quantum dots or organic/inorganic molecules for measuring signal from sandwich immunoassay. Such molecules are also either tagged directly to detection molecule in free form or after loading to other nanomaterials. Such tags on electrochemical oxidation/reduction provide the information of tags concentration which in turn can be related to the analyte concentration in the immunoassay.

Free Redox Marker and Redox Marker with Metallic Nanomaterial Based Enhancement
This section describes various different types of redox markers either in free form or loaded onto nanomaterials, investigated by researchers to enhance the sensitivity of sandwich-based immunoassays. The higher the presence of redox markers suggests higher responses, thus loading of such markers onto nanomaterials has shown promise in enhancing the sensitivity of immunoassays. Among various nanomaterials, AuNPs have gained maximum attention for achieving higher loading of redox tags. Yang et al. described the use of 6-ferrocenyl hexanethiol tagged AuNPs based probe development for PSA detection. With high physical loading of anti-PSA onto Fc tagged AuNPs, they were able to detect PSA down to 5.4 pg/mL [68]. Lin et al. introduced the use of AuNPs-mesoporous carbon form (MCF) as redox tag. In immunoassay physically tagged anti-CEA on Au/MCF was incubated with Ab1-antigen for 40 min and attached Au/MCF tags were then utilized for silver-deposition by incubating with enhancer solutions in dark for 4 min at 37 • C. Results of CEA detection using anodic stripping analysis revealed detection down to 0.024 pg/mL [71]. Chitosan-AuNP based detection probe was described by Chen and Ma. They utilized CHIT-PB-AuNP and CHIT-FC-AuNP probes for CEA and AFP detection, respectively. Corresponding Ab2 were physically immobilized on desired conjugate and used for immunoassay. For measurement, detection probe mixture was incubated with Ab1-antigen for 45 min at 37 • C and DPV signal was recorded in PBS. With this scheme, they were able to detect AFP and CEA down to 0.03 ng/mL and 0.02 ng/mL, respectively [52]. In another study, Feng et al. described the development of anti-AFP 2,2 -AuNPs-Thi@rGO and anti-CEA 2,1 -AuNPs-PB@rGO bioconjugates as detection probe. Probes were easily prepared by mixing and physical adsorption. For measurement, detection probes with physically adsorbed Ab2 were incubated with Ab1-antigen for 50 min at 37 • C and DPV measurements were carried out in PBS. Results indicate that with their probes, CEA and AFP can be estimated simultaneously down to 0.12 ng/mL and 0.08 ng/mL, respectively [36]. Figure 5 shows the preparation process of immunosensing probes. Further, Liu and Ma described PB-CS-Au and Cd-CS-Au based immunoprobes for CEA and AFP detection. With physically adsorbed Ab2, they were able to detect CEA and AFP simultaneously down to 0.006 ng/mL for AFP and 0.01 ng/mL for CEA [44]. Cd 2+ modified nanoporous TiO 2 has also been utilized for probe development for carbohydrate antigen 15-3 (CA15-3) detection down to 0.008 U/mL [32].
AFP can be estimated simultaneously down to 0.12 ng/mL and 0.08 ng/mL, respectively [36]. Figure 5 shows the preparation process of immunosensing probes. Further, Liu and Ma described PB-CS-Au and Cd-CS-Au based immunoprobes for CEA and AFP detection. With physically adsorbed Ab2, they were able to detect CEA and AFP simultaneously down to 0.006 ng/mL for AFP and 0.01 ng/mL for CEA [44]. Cd 2+ modified nanoporous TiO2 has also been utilized for probe development for carbohydrate antigen 15-3 (CA15-3) detection down to 0.008 U/mL [32]. Figure 5. Preparation process of immunosensing probes. In brief, reduced GO was first treated with Thi/Pb for their electrostatic binding, which was then utilized for physical adsorption of pre-synthesized AuNPs onto Thi/rGO or PDDA modified PB/rGO. The bound AuNPs were then utilized for physical adsorption of desired antibodies. Reproduced with permission from [36].
In a different strategy, polymer-nanotags based signal probes were described by Wang et al. for AFP and CEA detection. Probes were prepared by mixing metal ions (Cd 2+ , Pb 2+ ) modified Apo Figure 5. Preparation process of immunosensing probes. In brief, reduced GO was first treated with Thi/Pb for their electrostatic binding, which was then utilized for physical adsorption of pre-synthesized AuNPs onto Thi/rGO or PDDA modified PB/rGO. The bound AuNPs were then utilized for physical adsorption of desired antibodies. Reproduced with permission from [36].
In a different strategy, polymer-nanotags based signal probes were described by Wang et al. for AFP and CEA detection. Probes were prepared by mixing metal ions (Cd 2+ , Pb 2+ ) modified Apo solution with PLL-Au nanocomposites, which was then utilized for physical adsorption of Ab2. For detection via SWV, they utilized captured metal ions during immunoassay to deposit bismuth film at −1.2 V and estimated AFP and CEA at −0.78 V and −0.53 V, simultaneously. With this scheme they achieved detection down to 4 pg/mL for both targets [59]. Figure 6 shows the preparation process of immunosensing probes. Wang et al. also described the use of PtPNPs-Cd 2+ and PtPNPs-Cu 2+ hybrids based detection probes for immunosensing. Cd 2+ or Cu 2+ ions modified PTPNPs were utilized for physical immobilization of Ab2 and DPV signals were recorded for CEA and AFP at −0.736 V and 0.004 V, respectively. Using these probes and incubation for 1 h at 37 • C with Ab1-antigen conjugate, they achieved detection down to 0.002 ng/mL and 0.05 ng/mL for CEA and AFP, respectively [29]. Similarly, Wang et al. described the use of nanocubes of copper and cadmium hexacyanocobaltate based probes for CEA and AFP immunosensing and achieved detection down to 0.0175 ng/mL and 0.0109 ng/mL for CEA and AFP respectively [53]. solution with PLL-Au nanocomposites, which was then utilized for physical adsorption of Ab2. For detection via SWV, they utilized captured metal ions during immunoassay to deposit bismuth film at −1.2 V and estimated AFP and CEA at −0.78 V and −0.53 V, simultaneously. With this scheme they achieved detection down to 4 pg/mL for both targets [59]. Figure 6 shows the preparation process of immunosensing probes. Wang et al. also described the use of PtPNPs-Cd 2+ and PtPNPs-Cu 2+ hybrids based detection probes for immunosensing. Cd 2+ or Cu 2+ ions modified PTPNPs were utilized for physical immobilization of Ab2 and DPV signals were recorded for CEA and AFP at −0.736 V and 0.004 V, respectively. Using these probes and incubation for 1 h at 37 °C with Ab1-antigen conjugate, they achieved detection down to 0.002 ng/mL and 0.05 ng/mL for CEA and AFP, respectively [29]. Similarly, Wang et al. described the use of nanocubes of copper and cadmium hexacyanocobaltate based probes for CEA and AFP immunosensing and achieved detection down to 0.0175 ng/mL and 0.0109 ng/mL for CEA and AFP respectively [53]. Use of AuNPs modified mesoporous carbon CMK-3 has been described by Wu et al. to develop detection probes for CEA and SCCA estimation, simultaneously. For detection probe Au@CMK-3-anti-CEA-neutral red and Au@CMK-3-anti-SCCA-thionine conjugate were prepared via EDC/NHS chemistry and assay results using these probes showed detection down to 0.013 ng/mL and 0.010 ng/mL for CEA and SCCA, respectively [66]. Cu 2+ and Pb 2+ tagged AuNPs have also been used by Xu et al. for CEA and AFP detection down to 4.6 pg/mL and 3.1 pg/mL, respectively [54]. In other approach, Wang et al. described AuNPs modified mesoporous silica KIT-6 (Au@KIT-6) as surface to bind Ab2 (anti-CEA) and toluidine blue (TB) mediator based strategy for immunosensor development for CEA detection. With the developed probe (TB/Au@KIT-6/CMC/ILs-Ab2), they incubated Ab1-antigen complex for 1 h and achieved detection of CEA down to 3.3 fg/mL [45]. Figure 7 shows the preparation process of the immunosensing probes. Furthermore, they have shown that AuNPs modified multifunctional mesoporous silica (MCM-41) can be employed for Use of AuNPs modified mesoporous carbon CMK-3 has been described by Wu et al. to develop detection probes for CEA and SCCA estimation, simultaneously. For detection probe Au@CMK-3-anti-CEA-neutral red and Au@CMK-3-anti-SCCA-thionine conjugate were prepared via EDC/NHS chemistry and assay results using these probes showed detection down to 0.013 ng/mL and 0.010 ng/mL for CEA and SCCA, respectively [66]. Cu 2+ and Pb 2+ tagged AuNPs have also been used by Xu et al. for CEA and AFP detection down to 4.6 pg/mL and 3.1 pg/mL, respectively [54].
In other approach, Wang et al. described AuNPs modified mesoporous silica KIT-6 (Au@KIT-6) as surface to bind Ab2 (anti-CEA) and toluidine blue (TB) mediator based strategy for immunosensor development for CEA detection. With the developed probe (TB/Au@KIT-6/CMC/ILs-Ab2), they incubated Ab1-antigen complex for 1 h and achieved detection of CEA down to 3.3 fg/mL [45]. Figure 7 shows the preparation process of the immunosensing probes. Furthermore, they have shown that AuNPs modified multifunctional mesoporous silica (MCM-41) can be employed for detection probe development by immobilizing Ab2 and TB. Using such approach they detected AFP down to 0.05 pg/mL [57]. Metal alginate nanobeads (M-Alg), with different metals attached to specific detection antibodies can be employed for simultaneous estimation of biomarkers such as AFP, CEA and PSA [43]. Similarly Metal-Envision copolymer has also been utilized for detection probe preparation to achieve enhanced detection of Ca19-9, AFP and CEA [107].
Zhu et al. described the use of a hybridization chain reaction-based approach for testing four biomarkers simultaneously. For detection probe development, biotin-Ab2 was mixed with gold magnetic particles (Au/Sio 2 -Fe 3 O 4 ). The conjugate was then treated in sequence with streptavidin bio-S1, bio-S2 and bio-S3 for bio-dsDNA/SA/bio-Ab2/Au/SiO 2 -Fe 3 O 4 development via HCR reaction. Product was then modified with redox tag-streptavidin to obtain the detection probe. With such a probe they were able to detect AFP, CEA, CA125 and PSA down to 62, 48, 77 and 60 fg/mL, respectively [46]. A quadruple signal amplification strategy has been described by Zhou et al. for CEA detection. In amplification strategy streptavidin-labeled gold nanoparticles (AuNP-SA) were utilized for immobilizing detection antibody (Ab2) and initiator DNA strands (s0) using avidin-biotin coupling. For amplified electrochemical signal measurement, CEA sandwiched between Ab1 immobilized on sensor surface and modified Ab2 underwent hybridization with s1 and s2 DNA strands to form a concatamer followed by interaction with hemin, which resulted in formation of DNAzyme capable of binding with methylene blue. During DPV measurement, reduction of H2O2 by DNAzyme helped in enhancing methylene signal and sensor for CEA detection exhibited linearity in 1.0 fg/mL to 20 ng/mL range with detection limit of 0.5 fg/mL [60].
Zhang et al. described the development of a sensing strategy using signal tag of PtNP-ferrocenedicarboxylic acid based infinite coordination polymer (ICP) in combination with polyamidoamine dendrimers modified sensor electrode for PSA estimation in a sandwich type electrochemical ELISA. PtNP@ICP tag enhanced the catalytic reduction of H2O2 during the immunoassay to measure PSA. DPV measurements indicated that the sensor is able to detect PSA in the 0.001 to 60 ng/mL range with detection limit (LOD) of 0.3 pg/mL [61]. Shan   A quadruple signal amplification strategy has been described by Zhou et al. for CEA detection. In amplification strategy streptavidin-labeled gold nanoparticles (AuNP-SA) were utilized for immobilizing detection antibody (Ab2) and initiator DNA strands (s0) using avidin-biotin coupling. For amplified electrochemical signal measurement, CEA sandwiched between Ab1 immobilized on sensor surface and modified Ab2 underwent hybridization with s1 and s2 DNA strands to form a concatamer followed by interaction with hemin, which resulted in formation of DNAzyme capable of binding with methylene blue. During DPV measurement, reduction of H 2 O 2 by DNAzyme helped in enhancing methylene signal and sensor for CEA detection exhibited linearity in 1.0 fg/mL to 20 ng/mL range with detection limit of 0.5 fg/mL [60]. Zhang et al. described the development of a sensing strategy using signal tag of PtNPferrocenedicarboxylic acid based infinite coordination polymer (ICP) in combination with polyamidoamine dendrimers modified sensor electrode for PSA estimation in a sandwich type electrochemical ELISA. PtNP@ICP tag enhanced the catalytic reduction of H 2 O 2 during the immunoassay to measure PSA. DPV measurements indicated that the sensor is able to detect PSA in the 0.001 to 60 ng/mL range with detection limit (LOD) of 0.3 pg/mL [61]. Shan and Ma described the development of a multiple probe by attaching desired Ab2 with specific redox tag and utilized for simultaneous detection of five biomarkers. Using PBG-Au, PPP-Au, PTBO-Au, PMCP-Au and Cd NCs-based probes they detected CEA, NSE, CA125, Cyfra21-1 and SCCA simultaneously at 0.4 V, 0.15 V, −0.14 V, −0.5 V and −0.75 V in SWV scans and achieved detection down to 0.2 ng/mL for CEA, 0.9 ng/mL for NSE, 0.9 U/mL for CA125, 0.4 ng/mL for Cyfra21-1 and 0.03 ng/mL for SCCA [80]. Zhu et al. described the use of primer-AuNP-PSA aptamer-based probe with RCA reaction-based approach for enhanced detection of PSA. During immunoassay captured primer-AuNP-PSA aptamer was utilized for RCA reaction and CuNP formation in presence of sodium ascorbate and copper sulphate. Formed CuNPs were then extracted in HNO 3 and utilized for sensitive detection of PSA down to 0.02 fg/mL via DPSV measurements [83].

Redox Marker with Carbon Material Based Enhancement
Other than metallic nanomaterials, carbon materials in various forms such as graphene, CNTs, etc. have also gained much attention in enhancement strategies for electrochemical ELISA based assays. These materials provide support to load redox marker and detection antibody for signal enhancement. This section summarizes various such approaches described by researchers for enhancing the sensitivity of immunoassays. Using MWCNTs, Chen et al. described a AuNPs/SiO 2 @MWCNTs-based detection probe. For detection probe development, COOH-MWCNTs (c-MWCNTs) were first treated with PDDA to get positively charged MWCNTs, which were then treated with TEOS to make SiO 2 @MWCNTs. The obtained SiO 2 @MWCNTs were again treated with PDDA before incubating in AuNPs solution for 8 h to obtain a AuNPs/SiO 2 @MWCNTs nanocomposite. The composite was then incubated with thionine followed by an aptamer (Apt) solution, where Apt becomes covalently attached to AuNP via a thiol group. Using this probe, they were able to detect MUC1 down to 1 pM. Figure 8 illustrates the electrochemical sensing strategy for the detection of MUC 1, with the inset showing the preparation of Apt/Thi-AuNPs/SiO 2 @MWCNTs [58]. PtNPs modified graphene nanocomposites (PGN) were utilized by Jia et al. to develop detection probe for AFP and CEA detection. Using Fc-anti-AFP-PGN or Thi-anti-CEA-PGN they achieved detection down to 1.64 pg/mL and 1.33 pg/mL for CEA and AFP, respectively. Figure 9 illustrates the preparation procedure of PGN-Ab1/2 probes [48]. In another study, Li et al. described 3D graphene sheet (3DGS) prepared from GO reduction using NaI, based detection probe for CEA and AFP detection. On paper based assay with 3DGS@MB and 3DGS@Fc-COOH nanocomposites based probes resulted in CEA and AFP detection down to 0.5 and 0.8 pg/mL, respectively [76]. COOH-MWCNTs (c-MWCNTs) were first treated with PDDA to get positively charged MWCNTs, which were then treated with TEOS to make SiO2@MWCNTs. The obtained SiO2@MWCNTs were again treated with PDDA before incubating in AuNPs solution for 8 h to obtain a AuNPs/SiO2@MWCNTs nanocomposite. The composite was then incubated with thionine followed by an aptamer (Apt) solution, where Apt becomes covalently attached to AuNP via a thiol group. Using this probe, they were able to detect MUC1 down to 1 pM. Figure 8 illustrates the electrochemical sensing strategy for the detection of MUC 1, with the inset showing the preparation of Apt/Thi-AuNPs/SiO2@MWCNTs [58]. PtNPs modified graphene nanocomposites (PGN) were utilized by Jia et al. to develop detection probe for AFP and CEA detection. Using Fc-anti-AFP-PGN or Thi-anti-CEA-PGN they achieved detection down to 1.64 pg/mL and 1.33 pg/mL for CEA and AFP, respectively. Figure 9 illustrates the preparation procedure of PGN-Ab1/2 probes [48]. In another study, Li et al. described 3D graphene sheet (3DGS) prepared from GO reduction using NaI, based detection probe for CEA and AFP detection. On paper based assay with 3DGS@MB and 3DGS@Fc-COOH nanocomposites based probes resulted in CEA and AFP detection down to 0.5 and 0.8 pg/mL, respectively [76].    Xu et al. described the development of carbon and gold (CGN) nanocomposite-based immunoprobes for simultaneous detection of multiple cancer marker. CGN was prepared via glucose carbonization in the presence of sodium citrate followed by microwave reaction-based AuNPs deposition from HAuCl4. Further, redox tags were attached using reactive oxygen groups on CGN via mixing and stirring for 5 h. Physically immobilized antibody-based CGN-Thi-anti-CEA, CGN-DAP-anti-PSA and CGN-Cd 2+ -anti-AFP probes showed detection limit of 4.8, 2.7 and 3.1 pg/mL for PSA, CEA and AFP, respectively. Figure 10 illustrates the electrochemical probe development [75].  Figure 10 illustrates the electrochemical probe development [75].

Non-Enzymatic Catalytic Activity and Enzyme-Mimicking Materials Based Signal Amplification Strategies
The following section describes various alternative techniques other than those based on redox enzymes or tags to enhance the response of immunosensors. To get better sensitivity and more stable sensor systems, recently there has been a great interest in the development of detection probes, which can show non-enzymatic catalytic activity, or which can mimic enzymatic behavior. Among this category, various materials like Pt NPs, Fe3O4, etc. exhibiting catalytic activity towards H2O2 have gained maximum attention. In such approach, Cui et al. described mesoporous platinum nanoparticles (M-Pt NPs) as a non-enzymatic label-based immunosensor strategy. Ab2 were immobilized physically and during assay M-Pt showed high catalytic activity toward added H2O2 and the sensor exhibited detection limits of 7.0 pg/mL, 0.001 U/mL and 0.002 U/mL, for CEA, CA153 and CA125, respectively [30]. In another study, Feng et al. described a ferrocene modified ferroferric oxide@silica-amino groups (Fe3O4@SiO2-NH2)-based strategy for signal enhancement and detection of CEA.

Non-Enzymatic Catalytic Activity and Enzyme-Mimicking Materials Based Signal Amplification Strategies
The following section describes various alternative techniques other than those based on redox enzymes or tags to enhance the response of immunosensors. To get better sensitivity and more stable sensor systems, recently there has been a great interest in the development of detection probes, which can show non-enzymatic catalytic activity, or which can mimic enzymatic behavior. Among this category, various materials like Pt NPs, Fe 3 O 4 , etc. exhibiting catalytic activity towards H 2 O 2 have gained maximum attention. In such approach, Cui et al. described mesoporous platinum nanoparticles (M-Pt NPs) as a non-enzymatic label-based immunosensor strategy. Ab2 were immobilized physically and during assay M-Pt showed high catalytic activity toward added H 2 O 2 and the sensor exhibited detection limits of 7.0 pg/mL, 0.001 U/mL and 0.002 U/mL, for CEA, CA153 and CA125, respectively [30]. In another study, Feng et al. described a ferrocene modified ferroferric oxide@silica-amino groups (Fe 3 O 4 @SiO 2 -NH 2 )-based strategy for signal enhancement and detection of CEA.
For detection probe, Fe 3 O 4 particles prepared by a solvothermal method were first treated with TEOS to obtain Fe 3 O 4 @SiO 2 particles, which were then again treated with APTES to get Fe 3 O 4 @SiO 2 -NH 2 . For Fc-COOH and GA binding on prepared particles, Fc-COOH was first activated using EDC/NHS and then incubated with Fe 3 O 4 @SiO 2 -NH 2 and GA overnight with stirring. Fe 3 O 4 @SiO 2 /Fc/GA precipitates thus obtained were utilized for Ab2 binding by incubation at 4 • C for 2 h. During immunoassay, Fe 3 O 4 of captured probe provide catalytic activity towards H 2 O 2 , which in turn reduce Fc molecules and provide the detection signal. With such approach, they were able to detect CEA down to 0.0002 ng/mL. Figure 11 illustrates preparation of Fe 3 O 4 @SiO 2 -Fc-Ab2/HRP bioconjugate [35].

Non-Enzymatic Catalytic Activity and Enzyme-Mimicking Materials Based Signal Amplification Strategies
The following section describes various alternative techniques other than those based on redox enzymes or tags to enhance the response of immunosensors. To get better sensitivity and more stable sensor systems, recently there has been a great interest in the development of detection probes, which can show non-enzymatic catalytic activity, or which can mimic enzymatic behavior. Among this category, various materials like Pt NPs, Fe3O4, etc. exhibiting catalytic activity towards H2O2 have gained maximum attention. In such approach, Cui et al. described mesoporous platinum nanoparticles (M-Pt NPs) as a non-enzymatic label-based immunosensor strategy. Ab2 were immobilized physically and during assay M-Pt showed high catalytic activity toward added H2O2 and the sensor exhibited detection limits of 7.0 pg/mL, 0.001 U/mL and 0.002 U/mL, for CEA, CA153 and CA125, respectively [30]. In another study, Feng et al. described a ferrocene modified ferroferric oxide@silica-amino groups (Fe3O4@SiO2-NH2)-based strategy for signal enhancement and detection of CEA. For detection probe, Fe3O4 particles prepared by a solvothermal method were first treated with TEOS to obtain Fe3O4@SiO2 particles, which were then again treated with APTES to get Fe3O4@SiO2-NH2. For Fc-COOH and GA binding on prepared particles, Fc-COOH was first activated using EDC/NHS and then incubated with Fe3O4@SiO2-NH2 and GA overnight with stirring. A polyaniline-Au asymmetric multicomponent nanoparticles (PANI-Au AMNPs)-based strategy for immunosensor development was described by Fan et al. In their approach, captured PANI-Au AMNPs exhibited catalytic activity towards added H 2 O 2 and the sensor showed CA72-4 detection down to 0.10 U/mL. Figure 12 shows the schematic representation of the preparation of the PANI-Au AMNPs-Ab2 [55]. Gao et al. described the use of Pd-Au/C-based probe for SCCA detection. During the immunoassay, Pd-Au helped in achieving higher signal from H 2 O 2 during amperometric measurements and achieved detection down to 1.7 pg/mL [28]. The authors also proposed a Cu@Ag-CD-based enhancement strategy for immunosensor for CEA. In immunoassay Cu@Ag in H 2 O 2 presence generated an enhanced signal and achieved detection down to 20 fg/mL [37]. A palladium nanoparticles/carbon-decorated magnetic microspheres-based strategy for development of immunosensor for AFP was described by Ji et al. In this probe Fe 3 O 4 @C@Pd generated an enhanced signal for H 2 O 2 during amperometric measurement in the assay and achieved detection down to 0.16 pg/mL [33]. A Pt@CuO-MWCNTs based probe was described by Jiang et al. for AFP estimation. In presence of H 2 O 2 Pt@CuO-MWCNTs catalyzed the reaction and generated enhanced signal for AFP detection down to 0.33 pg/mL. Figure 13 shows the preparation procedures of Pt@CuO-MWCNTs/Ab2 [38]. of immunosensor for AFP was described by Ji et al. In this probe Fe3O4@C@Pd generated an enhanced signal for H2O2 during amperometric measurement in the assay and achieved detection down to 0.16 pg/mL [33]. A Pt@CuO-MWCNTs based probe was described by Jiang et al. for AFP estimation. In presence of H2O2 Pt@CuO-MWCNTs catalyzed the reaction and generated enhanced signal for AFP detection down to 0.33 pg/mL. Figure 13 shows the preparation procedures of Pt@CuO-MWCNTs/Ab2 [38].  Li et al. utilized nanoporous PtFe (NP-PtFe) alloys for probe development and enhanced catalytic conversion of H2O2 during amperometric measurement of CA15-3. With NP-PtFe, they achieved detection down to 3 × 10 −4 U/mL when measured in the presence of 5 mM H2O2 at −0.4V [31]. Li et al. described PdNi/N-GNRs-based probe for H2O2 catalysis. N-GNRs were prepared via microwave-assisted method and modified with PdNi. During assay PdNi enhanced catalysis and helped obtaining a higher signal for AFP detection. With such a probe they achieved detection down to 0.03 pg/mL when measured in the presence of 5 mM H2O2 [39]. Figure 14 shows the synthetic process of N-GNRs from N-MWCNTs and the synthetic process of PdNi/N-GNRs. In another study Li et al. described the use of Pb 2+ @Au@MWCNTs-Fe3O4 for enhanced H2O2 catalytic conversion for AFP detection down to 3.33 fg/mL [49]. Figure 15 illustrates the preparation procedure of Pb 2+ @Au@MWCNTs-Fe3O4/Ab2. of immunosensor for AFP was described by Ji et al. In this probe Fe3O4@C@Pd generated an enhanced signal for H2O2 during amperometric measurement in the assay and achieved detection down to 0.16 pg/mL [33]. A Pt@CuO-MWCNTs based probe was described by Jiang et al. for AFP estimation. In presence of H2O2 Pt@CuO-MWCNTs catalyzed the reaction and generated enhanced signal for AFP detection down to 0.33 pg/mL. Figure 13 shows the preparation procedures of Pt@CuO-MWCNTs/Ab2 [38].  Li et al. utilized nanoporous PtFe (NP-PtFe) alloys for probe development and enhanced catalytic conversion of H2O2 during amperometric measurement of CA15-3. With NP-PtFe, they achieved detection down to 3 × 10 −4 U/mL when measured in the presence of 5 mM H2O2 at −0.4V [31]. Li et al. described PdNi/N-GNRs-based probe for H2O2 catalysis. N-GNRs were prepared via microwave-assisted method and modified with PdNi. During assay PdNi enhanced catalysis and helped obtaining a higher signal for AFP detection. With such a probe they achieved detection down to 0.03 pg/mL when measured in the presence of 5 mM H2O2 [39]. Figure 14 shows the synthetic process of N-GNRs from N-MWCNTs and the synthetic process of PdNi/N-GNRs. In another study Li et al. described the use of Pb 2+ @Au@MWCNTs-Fe3O4 for enhanced H2O2 catalytic conversion for AFP detection down to 3.33 fg/mL [49]. Figure 15 illustrates the preparation procedure of Pb 2+ @Au@MWCNTs-Fe3O4/Ab2. Li et al. utilized nanoporous PtFe (NP-PtFe) alloys for probe development and enhanced catalytic conversion of H 2 O 2 during amperometric measurement of CA15-3. With NP-PtFe, they achieved detection down to 3 × 10 −4 U/mL when measured in the presence of 5 mM H 2 O 2 at −0.4V [31]. Li et al. described PdNi/N-GNRs-based probe for H 2 O 2 catalysis. N-GNRs were prepared via microwave-assisted method and modified with PdNi. During assay PdNi enhanced catalysis and helped obtaining a higher signal for AFP detection. With such a probe they achieved detection down to 0.03 pg/mL when measured in the presence of 5 mM H 2 O 2 [39]. Figure 14 shows the synthetic process of N-GNRs from N-MWCNTs and the synthetic process of PdNi/N-GNRs. In another study Li et al. described the use of Pb 2+ @Au@MWCNTs-Fe 3 O 4 for enhanced H 2 O 2 catalytic conversion for AFP detection down to 3.33 fg/mL [49]. Figure 15 illustrates the preparation procedure of Pb 2+ @Au@MWCNTs-Fe 3 O 4 /Ab2.    Au/Ag/Au core/double shell nanoparticles (Au/Ag/Au NPs) as novel enzyme-mimetic labels for anti-SCCA have been described by Wang et al. The study showed that improved electrocatalytic activity of Au/Ag/Au NPs for H2O2 reduction resulted in enhanced sensitivity and detection of SCCA down to 0.18 pg/mL [41]. For AFP estimation, Wei et al. described the use of anti-AFP tagged GO-CeO2 and Pd nanoparticle-based probes. In such a Pd/APTES-M-CeO2-GS-based probe, Pd octahedral NPs showed enhanced catalytic activity for H2O2 reduction and the sensor achieved detection down to 0.033 pg/mL when measured amperometrically at −0.4 V in the presence of 5 mM H2O2 [51]. Dumbbell shaped Pt-Fe3O4 as labels were described by Wu et al. for SCC estimation. Results indicate that Pt-Fe3O4 improve H2O2 reduction and the immunosensor achieved linearity in the 0.05-18 ng/mL range with a detection limit of 15.3 pg/mL [65]. In other study, Wu et al. described the use of a dumbbell-shaped PtPd-Fe3O4 nanoparticles-based label in designing immunosensor for CA72-4 biomarker for gastric cancer. Results indicated that PtPd-Fe3O4 improve H2O2 reduction and immunosensor achieved linearity in the 0.001-10 U/mL range with detection limit of 0.0003 U/mL [67]. Li et al. described the mesoporous core-shell Pd@Pt nanoparticles loaded by amino group functionalized graphene (M-Pd@Pt/NH2-GS)-based detection. With such a probe, they achieved higher reduction of H2O2 to improve the sensitivity of the immunosensor. In immunoassay for PSA Au/Ag/Au core/double shell nanoparticles (Au/Ag/Au NPs) as novel enzyme-mimetic labels for anti-SCCA have been described by Wang et al. The study showed that improved electrocatalytic activity of Au/Ag/Au NPs for H 2 O 2 reduction resulted in enhanced sensitivity and detection of SCCA down to 0.18 pg/mL [41]. For AFP estimation, Wei et al. described the use of anti-AFP tagged GO-CeO 2 and Pd nanoparticle-based probes. In such a Pd/APTES-M-CeO 2 -GS-based probe, Pd octahedral NPs showed enhanced catalytic activity for H 2 O 2 reduction and the sensor achieved detection down to 0.033 pg/mL when measured amperometrically at −0.4 V in the presence of 5 mM H 2 O 2 [51]. Dumbbell shaped Pt-Fe 3 O 4 as labels were described by Wu et al. for SCC estimation. Results indicate that Pt-Fe 3 O 4 improve H 2 O 2 reduction and the immunosensor achieved linearity in the 0.05-18 ng/mL range with a detection limit of 15.3 pg/mL [65]. In other study, Wu et al. described the use of a dumbbell-shaped PtPd-Fe 3 O 4 nanoparticles-based label in designing immunosensor for CA72-4 biomarker for gastric cancer. Results indicated that PtPd-Fe 3 O 4 improve H 2 O 2 reduction and immunosensor achieved linearity in the 0.001-10 U/mL range with detection limit of 0.0003 U/mL [67]. Li et al. described the mesoporous core-shell Pd@Pt nanoparticles loaded by amino group functionalized graphene (M-Pd@Pt/NH 2 -GS)-based detection. With such a probe, they achieved higher reduction of H 2 O 2 to improve the sensitivity of the immunosensor. In immunoassay for PSA detection they achieved detection down to 3.3 fg/mL [78]. Miao et al. described the use of PVP-stabilized colloidal iridium nanoparticles, prepared via ethanol reduction-based detection probe. Physically immobilized anti-CEA was used for detection and Ir NP-based catalyzed reduction of H 2 O 2 helped in achieving amperometric detection of CEA at −0.6 V down to 0.23 pg/mL [79]. Tables 2 and 3 show the details of various strategies used for development of detection probes for enhanced detection and the various characteristics of the developed immunoassays using electrochemical ELISA, respectively.

Conclusions and Outlook
In the last few years, researchers have shown that electrochemical ELISA-based immunosensors can achieve similar or even better performance when compared to traditional optical ELISA immunoassays and are capable of replacing them in the near future. The innovations in nano-and bio-technologies and in surface and material chemistry have resulted in the development of novel sandwich assays with improved performance and stability. Further, due to the use of electrochemical techniques for testing, they have the advantage of providing faster response and on site testing in either undiluted or treated samples. This review has also described the various approaches which have been attempted by researchers to develop novel electrochemical immunosensors. It is clear that newer matrices and immobilization platforms allow higher capturing molecule loading and thus enhanced signals. Furthermore, the use of carrier materials for detection tags before electrochemical measurement helps in enhancing the sensitivity of the immunosensors. Although there are many success stories, there are a few limitations which need further detailed investigation before these electrochemical ELISA-based sensors can be accepted in clinical practice and able to replace optical ELISA. To improve the shelf life of the systems and to improve their stability, more detailed research is still required to understand the nature of biomolecule bound on flat matrices and on nanomaterials. Further, more studies are required for better surface blocking to prevent non-specific binding, while maintaining conductivity of sensor surface for higher electrochemical signal and better sensitivity. Moreover, newer and better packaging approaches are required to be developed to hold all the required chemicals and reagents required in a multistep ELISA processor, so that the assay can be automated and made less prone to human errors. It is envisioned that further advancements in nano-and bio-technology along with chemistry, material science, physics and electronics will pave the way to solve these issues and result in larger acceptance of these devises in clinical practice.