Affinity biosensors for tumor-marker analysis.

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Affinity biosensors for tumor-marker analysis

The use of cancer biomarkers is emerging as one of the most promising strategies for early detection and management of cancer. Biosensors can provide advanced platforms for biomarker analysis with the advantages of being easy to use, inexpensive, rapid and offering multi-analyte testing capability. The intention of this article is to discuss recent advances and trends in affinity biosensors for cancer diagnosis, prognosis and even theragnosis. The different types of affinity biosensors will be reviewed in terms of molecular recognition element. Current challenges and trends for this technology will be also discussed, with a particular emphasis on recent developments in miRNA detection.

Even if overall cancer death rates continue to decrease in the United States [1] and Europe [2] , cancer is still a leading cause of death. In 2014, it is estimated that there will be 1,665,540 new cases of all cancer sites and an estimated 585,720 people will die of this disease in United States, as reported by the Surveillance Epidemiology and End Results Program of the National Cancer Istitute. In 2012 in Europe, there were just over 3.4 million new cases of cancer (excluding nonmelanoma skin cancers) [3] , and the estimated total number of cancer deaths was 1.75 million, of which 56% were in men and 44% in women. Cancer is not just one disease but many diseases. There are more than 100 different types of cancer affecting over 60 human organs. Following statistics, in Europe in 2012 the most common primary cancer sites in men were prostate, lung, colorectal and bladder. In women, breast cancer was by far the most frequently diagnosed neoplasm, followed by colorectal, lung and corpus uteri cancers. Lung cancer, with an estimated 353,000 deaths (a fifth of the total) was the most frequent cause of death from cancer in Europe in 2012, followed by colorectal cancer, breast cancer and stomach cancer [3] . In cancer, genetic and epigenetic alterations can cause the onset of the disease and

10.4155/BIO.14.247 © 2014 Future Science Ltd

Ilaria Palchetti Dipartimento di Chimica, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (Fi), Italy [email protected]

the formation of cancer cells which will exhibit higher growth capability than normal mammalian cells. Unlimited cell division, invasion of nearby tissues, metastasis to distant organs, resistance to antigrowth signaling, evasion of apoptosis and sustained angiogenesis are typical features of cancer. Mutations arising in somatic cells account for most cancers and are frequently caused by many environmental factors such as the exposure to carcinogenic chemicals, hormones, radiations, bacterial or viral infections. Many other factors in diet and lifestyle may cause or prevent cancer. The genes that are altered in somatic cells to cause cancer are referred to as oncogenes or tumor suppressor genes. Oncogenes, when mutated or modified, stimulate abnormal cell survival and division. Tumor suppressor genes are lost in cancer resulting in loss of control of cell growth. Genetic alterations can occur at different levels. Changes in single bases within the DNA sequence, DNA replication errors that add or delete bases, malfunction of the DNA mismatch repair genes or deletion or amplification of DNA sequences (ranging from short sequences to large portions of chromosomal arms) are examples of genetic alterations that can lead to cancer initiation. In addition, many

Bioanalysis (2014) 6(24), 3417–3435

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Key terms microRNA: microRNAs are an important class of short, noncoding (approximately 22 nucleotides in length) endogenous RNAs Biosensor: A biosensor is a compact analytical device, capable of providing specific quantitative or semiquantitative analytical information using a biological or biologically derived recognition element, which is in direct spatial contact with a transducer element. Cancer biomarker: Cancer biomarkers are molecular changes that can be detected in the tumor or in the blood, urine, or other body fluids of cancer patients.

chromosomal changes (i.e., increased or decreased number of chromosomes and chromosomal rearrangements) have been identified in cancer cells. Epigenetic alterations are modifications of DNA that affect gene expression without altering DNA nucleotide sequences, such as DNA methylation, histone modifications or nucleosome positioning. Modifications in noncoding RNAs expression, specifically in microRNA expression, are other examples of epigenetic alterations [4] . Cancers are described by their pathological grade and clinical stage. Knowing the stage of disease helps the doctor plan treatment and estimate the person’s prognosis. Historically, the most important cancer diagnostic and prognostic indicators are morphological and histological characteristics of tumors. The development of molecular profiling technologies to produce ‘molecular signatures’ provides the potential to tailor medical care, both at tumor and patient levels. Delivering the right dose for the right indication to the right patient at the right time [5] might be possible using these approaches. Personalized cancer therapy allows increasing therapeutic efficacy while at the same time decreasing inadvertent toxicity [5] . Biosensors can enter this scenario providing specific, easy-to-use, cost-effective and rapid diagnostic tools [6–9] . Herein, the main features, the current challenges and future trends of biosensor technology for cancer biomarker detection will be discussed. The different types of affinity biosensors will be reviewed in terms of molecular recognition element. Cancer biomarker: definition & general considerations As reported by the National Cancer Institute (NCI) dictionary of cancer terms, “A biomarker is a biological molecule found in blood, other body fluids or tissues that is a sign of a normal or abnormal process, or of a condition or disease.” Cancer biomarkers encompass a wide variety of molecules including proteins (e.g., enzymes, antibodies or receptors), nucleic acids (NAs), hormones and

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peptides. A biomarker can also be a collection of alterations, such as gene expression, proteomic and metabolomic signatures [10] . Biomarkers can be found in blood (either total blood, serum or plasma) or other biological fluids like urine or sputum, and thus easily assessed, or can be tissue-derived and require either biopsy or special imaging for evaluation. Biomarkers can be used in multiple clinical settings, not only to identify the presence of a tumor but also to estimate the risk of disease or to screen for occult primary cancers, to distinguish benign from malignant findings or cancer status and subtype, to detect recurrence or determine response to therapy [10,11] . A list of some biomarkers and examples of their role in clinical settings are given in Table 1. Some biomarkers are only used in a specific setting, whereas others can serve more than one purpose. Recently, most of the attention concerning molecular cancer diagnostics in the clinical setting has been focused on predictive biomarkers of response to therapy. Indeed, the success of personalized cancer therapy depends on having accurate diagnostic tests that identify patients who can benefit from targeted therapies [11,12] . A classic example is that of breast cancer: clinicians now commonly use diagnostics to determine which breast tumors overexpress the HER2, which is associated with a worse prognosis but also predicts a better response to the medication trastuzumab [13] . Tests for HER2 and other biomarkers are approved along with the drugs (as ‘companion diagnostics’) so that clinicians can better target patient’s treatment. Moreover, differential diagnosis or evaluation of recurrence of the disease is another important clinical application of molecular diagnostics in oncology. For example, molecular analysis of softtissue sarcomas is emerging as a critical tool for their differential diagnosis. Such analysis includes SS18SSX, EWSR1 and PAX3/7- FKHR fusion proteins in synovial sarcomas, Ewing’s sarcoma, alveolar rhabdomyosarcomas, respectively [12,13] . Another important aspect of molecular diagnostics is the analysis of prognostic markers in certain malignancies such as the recurrence risk stratification using the OncotypeDx and Mammaprint gene expression signatures in breast cancer, or the IHC4 immunohistochemistry method that measures the expression of the estrogen receptor, the progesterone receptor, human EGFR2/HER2 and Ki-67 [12] . The emerging need in cancer diagnostics is to move away from single biomarker analysis. It is necessary, instead, to perform ‘panel testing’ for a variety of biomarkers on which predictive, diagnostic, therapeutic and prognostic assessment may be based. Some examples of multi-parameter assays have recently emerged, in which multiple analytes, such as expression of sev-

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Table 1. Some biomarkers of breast and prostate cancer diseases with their role in clinical settings. The complete list of biomarkers can be found at NCI biomarker website. Biomarker

Cancer types

Tissue analyzed Role in clinical setting

CA15–3/CA27.29

Breast cancer

Blood

To assess whether treatment is working or disease has recurred

Calcitonin

Medullary thyroid cancer and breast cancer

Blood

To aid in diagnosis, check whether treatment is working, and assess recurrence

CEA

Colorectal cancer and breast cancer

Blood

To check whether colorectal cancer has spread; to look for breast cancer recurrence and assess response to treatment

ER/PR

Breast cancer

Tumor

To determine whether treatment with hormonal therapy (such as tamoxifen) is appropriate

HER2/neu

Breast cancer, gastric cancer and esophageal cancer

Tumor

To determine whether treatment with trastuzumab is appropriate

PSA

Prostate cancer

Blood

To help in diagnosis, assess response to treatment and look for recurrence

uPA and PAI-1

Breast cancer

Tumor

To determine aggressiveness of cancer and guide treatment

21-Gene signature (Oncotype DX)

Breast cancer

Tumor

To evaluate risk of recurrence

70-Gene signature (Mammaprint)

Breast cancer

Tumor

To evaluate risk of recurrence

CEA: Carcinoembryonic antigen; ER: Estrogen receptor; PAI: Plasminogen activator inhibitor; PR: Progesterne receptor; PSA: Prostate-specific antigen; uPA: Urokinase plasminogen activator.

eral genes, are measured, and a weighted algorithm is developed to generate a single result, index, signature or ‘score,’ like in OncotypeDx or Mammaprint, etc. [14] . Given the increasingly critical role of molecular analysis in cancer patient management, there is a clear need for developing robust, high-quality diagnostic tests and for their corresponding technical and clinical validation [12] . Thorough technical validation is a prerequisite for establishing the performance characteristics of a methodology. Evaluation of sensitivity, specificity, limit of detection (LOD) and accuracy should be included as part of a standardized framework for the validation and verification of clinical molecular tests. Affinity biosensors as diagnostic tools in cancer Biosensors are analytical devices incorporating a biological sensing element. They harness the exquisite sensitivity and specificity of biology in conjunction with physicochemical transducers to deliver complex bioanalytical measurements with simple, easy-to-use, cost-effective formats [15] . Biosensors are mainly classified according to the biological specificity conferring mechanism (catalytic


or affinity biosensors) or, alternatively, to the mode of physical-chemical signal transduction. Catalytic systems integrate enzymes, tissues or whole cells with transducers that act as biological catalysts for particular reactions. On the contrary, affinity biosensors, take advantage of different biological elements, such as antibodies, receptors or nucleic acids (NAs). In affinity biosensors, the binding between the target analyte and the immobilized biomolecule on the transduction element is governed by an affinity interaction under thermodynamic considerations, like the antigen-antibody (Ag-Ab), the DNA hybridization or the protein-NA binding. Regarding the transduction principles, biosensors can be classified as optical, electrochemical, mass, magnetic, calorimetric or micromechanical biosensors. Optical detection by fluorescence spectroscopy is a popular method, due largely to the ease with which biomolecules can be fluorescently labeled, the availability of many different fluorophores and quenchers [16] , and the inherent capability for real-time multiplex detection. Chemiluminescence is another optical technique widely used [17] . A different optical transduction, based on evanescent wave device, can offer real-time

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Review Palchetti label-free optical detection. These biosensors rely on monitoring changes in surface optical properties (shift in resonance angle due to change in the interfacial refractive index) resulting from the surface-binding reaction [18] . Electrochemical devices have also proved to be very useful, due to their inherent miniaturization, cost and compatibility with advanced microfabrication technology. Electrochemical detection usually involves monitoring a current response under controlled potential conditions. However other changes in electrochemical parameters such as capacitance, impedance and conductivity have been used [19,20] . In photoelectrochemical measurements, light is used to excite active species on the electrode and photocurrent is obtained as the detection signal. This technique has particularly benefited by the emergence of nanomaterials with enhanced photoelectrochemical properties. Quartz crystal microbalance transducers, comprising an oscillating crystal, can be used in label-free detection schemes, since the increased mass, associated with the biorecognition reaction, results in a decrease of the oscillating frequency. Micromechanical transduction is based on cantilevers; in this case biomolecular interactions can be detected via the bending of microfabricated cantilevers coated with bioreceptors. Finally, in magneto-biosensor, magnetic labels are used to detect magnetoresistance, giant magnetoresistive effect, spin-value giant magnetoresistive effect and other parameters. Biosensor technology has deeply benefited from the recent advances in nanotechnology. Several areas including transducers, labeling technology and manufacturing have been implemented by nanotechnology [21,22] . The use of nanomaterials may increase the sensitivity of a biosensor. Moreover, new detection schemes have been developed employing nanomaterials. For example, when metal nanoparticles, such as gold nanoparticles (AuNPs), modified with specific ligand interact with the target (i.e., a protein) the resulting nanoparticle aggregation may be measured by light absorption. AuNPs can also be used to scatter light of specific wavelengths. When functionalized AuNPs bind specific target proteins, the scattered light intensity or wavelength will change depending on the NP size, composition and on the degree of surface plasmon resonance (SPR). Moreover, miniaturization to nanoscale size, reduced cost of production, high throughputs are other examples of the advantages offered by nanotechnology. Some achievements in the use of nanoparticles to detect cancer biomarkers and cancer cells are well reviewed by Perfezou et al. [22] . Examples of optical, mechanical and magnetic nanosensors are recently reviewed by Swierczewska et al. [21] . The increasing number of publications over the last 10 years highlights the interest in biosensors, as diag-

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nostic tools in cancer. Biosensors have several potential advantages over other methods of cancer analysis, especially specificity, increased flexibility, reduced costs and assay speed. In addition, biosensors can allow multi-target analyses, automation and reduced costs of testing. Biosensor-based diagnostics might facilitate cancer screening, improve the rates of earlier detection and facilitate a clear decision structure for patient management and prognosis. There are thousands of examples of affinity biosensors for cancer biomarker detection and this article cannot obviously review all. In this article, recently reported affinity biosensors, mainly within the past 5 years, will be reviewed. Moreover, since molecular recognition is a central event for most biosensors, this review will focus on the recent advances in designing the molecular recognition elements. The molecular recognition element The past few years have seen continued emphasis on antibody-based and hybridization-based biosensors. However, the emergence of new recognition approaches, most notably those based on aptamers and peptides, is bringing new perspectives in biosensor development. Interesting alternative approaches are, also, those based on phages, molecular imprinted polymers and carbohydrate affinity binding. These ligands are reviewed in the following sections. Antibody-based biosensors

Antibody-based biosensors, also called immunosensors, are inherently versatile in that antibodies specifically bind to individual compounds or groups of structurally related compounds with a wide range of affinities. Antibodies are the most used affinity proteins for all life science applications. They can be isolated to many targets with high affinity and specificity. The most used antibody type, the IgG molecule, is a 150 kDa protein composed of four polypeptide chains, two identical larger heavy chains and two identical shorter light chains [23] . A huge number of articles dealing with the development of antibody-based biosensor (or immunosensor) for cancer biomarker detection are reported in literature, confirming that antibodies are still one of the mainstay affinity elements. The main antibody formats used in immunoassays are polyclonal and monoclonal antibodies. Some examples of immunosensors for the detection of biomarkers using antibodies, as biomolecular recognition element, coupled to different transducing principles are reported in Tables 2 & 3. In particular, examples concerning the detection of carcinoembryonic antigen (CEA) and examples of biosensor formats



for the prostate-specific antigen (PSA) detection are reported in Tables 2 & 3, respectively. CEA is a highly glycosylated cell surface glycoprotein, most often associated with colorectal cancer but also found at elevated levels in patients with breast cancer, ovarian cancer and lung cancer. The normal blood serum concentration of CEA in healthy individuals is less than 2.5 ng/ml, with increased levels suggesting the presence of a tumor. PSA is a commonly used but controversial biomarker for screening prostate cancer. A normal level of PSA is 4.0 ng/ml. PSA level between 4.1 and 9.9 ng/ml suggests the presence of prostate cancer. However, in addition to prostate cancer, elevated PSA levels may also indicate benign prostatic hyperplasia, prostatitis (inflammation of the prostate), or smaller tumors that do not prove to be fatal. Following its approval by the US FDA in 1986, increased screening of men over age 50 led to an increase in the diagnosis of prostate cancer, but there were concerns raised about overtreatment [10] . While antibodies are excellent ligands in terms of affinity and selectivity, they are expensive to produce, incompatible with some high-throughput approaches and often have limited shelf life [6] . Significant prog-

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ress has been made in developing stable recombinant antibody fragment libraries [24] ; however, valuable alternatives are still required. Nanobodies (Nbs) are single-domain antibody (sdAb) fragments formed by the variable domain of heavychain-only antibodies (HCAbs) found in camelids (e.g., dromedaries, llamas and alpacas) also known as VHH (for VH of HCAbs) [25] . However, similar functional antibodies devoid of L chains are also present in an assortment of nonmammals, such as sharks (Orectolobus maculates and Ginglymostoma cirratum) and ratfish [26] . In Figure 1 is reported a scheme of these immunomolecules. These sdAbs constitute the smallest domains of natural Abs having full antigen-binding capacity (molecular weights ranging between 12 and 14 kDa) and have demonstrated to be an advantageous alternative to monoclonal antibodies (mAbs). Nbs can be encoded by a single VHH gene segment and are produced in bacteria and yeast with less cost than conventional mAbs. Nbs are reported to be particularly suited for the development of biosensor because site-specific functional groups are easily introduced, allowing covalent and oriented binding with minimal loss of specific-

Table 2. Some examples of recent carcinoembryonic antigen immunosensor platforms. Transducer

Label/label-free

Detection limit

Ref.

SPR

Direct and sandwich assay

3 ng/ml

[105]

SPR

Au Nps

0.1 ng/ml

[106]

Electrochemical

Nanotubular mesoporous PdCu alloy

4.86 pg/ml

[107]

Electrochemical

AuPt nanochains – HRP

0.11 pg/ml

[108]

Electrochemical

Double-strand DNA-Au NP tag and hexaammineruthenium (III) chloride

3.2 fg/ml

[109]

Electrochemical

Label free

5 ng/ml

[110]

Electrochemical

Enzymatic label

0.2 ng/ml

[111]

Electrochemical

Ferrocene and HRP

0.01 ng/ml

[112]

Electrochemical

Nanogold magnetic particle

0.001 ng/ml

[113]

Electrochemical

Multiplexed Microfluidic device

0.5 pg/ml

Electrochemical

Label free

0.015 fg/ml

Electrochemical

Label free

6 fg/ml

[115]

Electrochemical

Au-ZnO nanoparticles

0.2 ng/ml

[116]

Electrochemical

Nanogold-enwrapped graphene nanocomposites

0.1 ng/ml

[117]

Electrochemical

Prussian Blue nanoparticles coated with poly(diallyldimethylammonium chloride) and double-layer gold nanocrystals

0.2 ng/ml

[118]

Photoelectrochemical

Multiwalled carbon nanotube-Congo redfullerene nanohybrids

0.1 pg/ml

[119]

Electrochemiluminescent

Label based: QD

0.4 pg/ml

[120]

Magnetic

Label based: magnetic beads

n.d.

[121]

[95] [114]

AUNP: Gold nanoparticle; HRP: Horseradish peroxidase; N.d. Not determined; QD: Quantum dots; SPR: Surface plasmon resonance.



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Review Palchetti ity and affinity [27] . The Nb small size provides highbinding capacity surfaces resulting in higher sensitivity, and the high stability and refolding competence of Nbs allow stringent washing and regeneration conditions. A PSA immunosensor based on a commercially available SPR chip and an sdAb, cAbPSAN7, derived from dromedary heavy-chain antibodies and identified after phage display is reported in [28] . Although PSA concentrations as low as 10 ng/ ml could be detected directly, this detection limit could be enhanced to PSA levels in the sub nanogram per milliliter range by introducing a sandwich assay involving a biotinylated secondary antibody and streptavidin-modified gold nanoparticles. Recently, Campuzano and Pingarron [29] described the development of two amperometric immunosensors for the determination of fibrinogen (Fib), a biomarker of bladder cancer, in human plasma involving a novel synthesized specific Nbs. The assay implied the use of disposable screen-printed electrodes and functionalized magnetic beads as solid supports to perform the immunological reactions. Two different competitive magnetoimmunosensing configurations (direct and indirect) were developed and their applicability compared. The immunosensor was successfully employed for the analysis of a commercial plasma sample with certified Fib content. Furthermore, in order to find alternatives to antibodies, different and very promising avenues of investigation have been pursued including NA aptamers and artificial combinatorial proteins and peptides. The next two paragraphs are dealing with these topics.

Aptamer-based biosensors

Aptamers are single-stranded DNA or RNA ligands, which can be selected for different targets starting from a huge library of molecules containing randomly created sequences. The selection process is called systematic evolution of ligands by exponential enrichment (SELEx). The SELEx process involves iterative cycles of selection and amplification starting from a large library of oligonucleotides with different sequences (generally 1015 different structures). The number of cycles required depends on the stringency conditions, but once obtained and once the sequence is known, unlimited amounts of the aptamer can be easily achieved by chemical synthesis. In addition to this very important aspect of having an unlimited source of identical affinity recognition molecules available, aptamers can offer advantages over antibodies that make them very promising for bioanalytical applications [30] . The main advantage is the overcoming of the use of animals or cell lines for the production of the molecules. The aptamer selection process is faster than other methods and can be manipulated to obtain aptamers that bind a specific region of the target and with specific binding properties in different binding conditions. After selection, aptamers are produced by chemical synthesis and purified to a very high degree by eliminating the batch-to-batch variation found when using antibodies. By chemical synthesis, modifications in the aptamer can be introduced enhancing the stability, affinity and specificity of the molecules. Several aptamers have already been developed against cancer-related proteins, like CEA [31] , and have been adapted for numerous biosensing principles

Table 3. Some examples of recent prostate-specific antigen immunosensor platforms. Transducers

Label/label-free

Detection Limit

Ref.

SPRi

Quantum dots

100 pg/ml

[122]

Electrochemical

Label-based: graphene sheet sensor platform and ferrocene functionalized iron oxide nanoparticle

2 pg/ml

[123]

Electrochemical

Logic based device

n.d.

[124]

Electrochemical

Sandwich assay

5 pg/ml

[125]

Electrochemical

Sandwich assay based on magnetic beads

1.7 ng/ml

[126]

Electrochemical

Sandwich assay based on graphene and HRP

1 pg/ml

[127]

Field Effect Transistor

Label-free: graphene oxide

100 fg/ml

[128]

Colorimetric

Paper-based colorimetric biosensing platform

n.d.

[129]

Magnetic

Label free

100 pg/ml

[130]

Piezoelectric

Label free

0.25 ng/ml

[131]

SPR and Quartz Crystal Microbalance

Label based: Au nanoparticles

0.29 ng /ml

[132]

8 pg/ml

[133]

Electrochemiluminescence Sandwich assay using glucose oxidase and luminol HRP: Horseradish peroxidase; N.d.: Not determined; SPRi: Surface plasmon resonance imaging.

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A

B

Nb

VL

V

H

scFv

Review

V H

H

V

H

H

V H

V

VL

CH2

CH2

Fc

CH3

Fc

C

D

sdAb

R

C3

C3

C3

C4

C4

C4

C5

C5

C5

C6

C6

C6

C4

C3 C5 C6

C2

N V-

A

L

C

CL

C2

AR

N V-

1

C

CL

C2

L

V

VL

V

H

H

V

AR

N V-

C2

CH2 CH3

CL

CH3

CH2

L

CH3

H

1

H

C C

Fab

VH

H

L V

1 CH

Figure 1. Scheme of immuno-molecules in different animal species. (A) Conventional mammalian IgG antibodies; (B) camelid heavy chain-only antibodies; (C) cartilaginous fish IgW and, (D) shark Ig-NARs. The smallest practical antigen-binding entity of IgG, the scFv, is also shown, as well as the single-domain antigen-binding fragments of the camelid heavy chain only antibodies and shark Ig-NAR, known as nanobody and V-NAR (sdAb), respectively. C: Constant domain; CH: Constant domain of the heavy chain; CL: Constant domain of the light chain; Nb: Nanobody; sdAb: Single-domain antibody; VH: Variable domain of the heavy chain; VHH: N-terminal variable domain of the heavy chain-only antibody; VL: Variable domain of the light chain; V-NAR: Variable antigen-binding domain. Reprinted with permission from [23] © Elsevier (2009).



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Key term Circulating tumor cells: Tumor cells that escape from the primary tumor into the bloodstream and travel through the circulatory system to distant sites where they develop into secondary tumors.

mainly in direct and sandwich formats than in competitive formats. Excellent reviews have been published, covering the application of aptamer biosensing in medicine studies [30,32–35] , and we ask the reader to refer to these reviews to find more examples of the different sensing procedures. In recent years, aptamer-based biosensors have been frequently proposed for monitoring cancer cells in peripheral blood. Tumor cells that escape from the primary tumor into the bloodstream and travel through the circulatory system to distant sites where they develop into secondary tumors are called circulating tumor cells (CTCs). These are the cells that cause metastasis. The discovery of CTCs is considered as a milestone in cancer research that is shedding light on the mechanisms of cancer progression. During the last decade, there has been considerable discussion about monitoring CTCs for the purpose of early cancer detection and prediction of treatment outcome. However, the potential use of CTCs as prognostic markers is still controversial [36] . The selection procedure known as cell-SELEx [37] , and its refinements [38] , is particularly useful for the identification of aptamers that are able to bind to their cognate target on the cell surface membrane. Aptamers evolved by cell-SELEx have been conjugated with suitable labels [39] for the different transducing principles. For example, gold nanoparticles have been used to develop a colorimetric assay [40] , able to detect a minimum of 800 cells with a portable strip reader within 15 min in human blood. Moreover, many electrochemical [41,42] , photoelectrochemical [43] and electrochemiluminescent [44] biosensor formats have also been used for the detection of CTCs. Engineered protein-based & peptide-based biosensors

The increasing experience in the field of combinatorial libraries and protein engineering has inspired researchers to develop new nonimmunoglobulin affinity protein without the limitations of antibodies. Consequently, today antibodies are facing increasing competition from a large number of so-called engineered protein scaffolds [30] . There are presently approximately 50 suggested protein scaffolds reported and these have been intensely reviewed over the last years [45,46] . Example of nonimmunoglobulin proteins that can be used in biosensors are the Affibody ® molecules, based on the immunoglobulin binding B

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domain of protein A from the bacteria Staphylococcus aureus [45] . Affibody molecules able to recognize HER2 or TNF-α have been selected and are already commercially available. Furthermore, many peptide ligands that are able to target specific cancer biomarkers have been selected [47] and used in biosensors [48,49] . Methods that have been developed to identify peptide ligands include phage display libraries, synthetic library methods requiring deconvolution, one-bead-one-compound combinatorial libraries and others. Phage display technology is a powerful technique for ligand selection. Peptides have been isolated that target tumor-associated molecules such as receptors, vasculature components, lipids and carbohydrates. Examples of phage display derived tumor targeting peptides are described by Deutscher et al. [47] . Peptides can be chemically synthesized in large quantities once the optimal peptide with the required affinity has been identified. However, peptides have relatively lower affinity when compared with antibodies. Therefore designing the assay is important for developing successful sensing methods when incorporating peptides in biosensors [7] . Research in the development of higher affinity peptide as ligands is still in evolution [47] . Bacteriophage-based biosensors

Bacteriophages (phages) are traditionally used for the development of phage display technology. Recently, not only have the peptides displayed on phage been employed, but the phage themselves have been used in biosensing [50,51] with a number of transducer platforms. Furthermore, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight toward their use for in vivo imaging and sensing [51,52] . Regarding biosensor development, phages can be grouped into the following categories regardless of their filamentous or icosahedral classification (Figure 2) [50,53] : nonlytic phages (i.e., M13) displaying the target-specific peptides or proteins that can be used as a target-recognizing sensing probe as antibody substitutes; phages to be used in phagedisplay to select target-recognizing peptides, proteins or antibodies that can be chemically synthesized or genetically produced, and directly used as probes; lytic phages (T4 or T7) that can act as bacteria-sensing probes by breaking its bacteria host strain specifically and releasing the cell-specific contents from the bacteria, thereby leading to the detection of the specific bacteria strain; and the phage nanofibers that can be conjugated with other organic or biological molecules and/or assembled with other nanomaterials to form a composite device that can be respon-



sive to some external stimuli (nanoscaffolding material). Examples of phage-based biosensors for cancer biomarkers are reported in literature [53] . Molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) are an interesting class of synthetic molecules for biosensor development. MIPs are synthetic polymeric materials with specific recognition sites complementary in shape, size and functional groups to the template molecule, involving an interaction mechanism based on molecular recognition. These recognition sites mimic the binding sites of biological entities such as antibodies. In particular, in MIPs production, functional and cross-linking monomers are copolymerized in the presence of a target analyte (the imprint molecule), which acts as a molecular template. The imprint molecule, initially, forms a complex with the functional monomers, and after polymerization, their functional groups are held in position by the highly cross-linked polymeric structure. Subsequent removal of the imprint molecule reveals binding sites that are complementary in size and shape to the analyte. Due to their synthetic nature, their stability, ease of preparation and low cost, MIPs have found numerous analytical applications including sensing. MIPs can be prepared according to a number of approaches that are different in the way the template is linked to the functional monomer and subsequently to the polymeric-binding sites. Thus, the template can be linked and subsequently recognized by reversible covalent bonds, metal ion coordination or noncovalent bonds. The covalent approach is interesting because of the higher stability of the prepolymerization complex. However, the most widely applied technique to generate molecularly binding sites is based on noncovalent self-assembly of the template with functional monomers prior to polymerization with cross-linking monomer and then template extraction followed by rebinding via noncovalent interactions [54,55] . Different methods used for developing proteinimprinted polymers and their incorporation with a number of transducer platforms with the aim of identifying the most promising approaches for the preparation of MIP-based protein sensors have recently been reviewed [54,55] . Recently Wang et al. developed sensing elements for the detection of CEA (detection range 2.5–250 ng/ml) [56] , while Viswanathan et al. [57] developed a protein-imprinted polymer for the electrochemical detection of epithelial ovarian cancer antigen-125 (CA 125), reporting a detection limit of 0.5 U/ml. Sugar-based biosensors

The surface of a cell comprises thousands of different lipids, proteins and carbohydrates. Furthermore, many membrane lipids and proteins are conjugated to


Review

polysaccharides, which comprise the glycocalyx of all cells. The cell surface glycan expression is considered a good candidate marker to distinguish the different expression profiles during a variety of cell biological processes [58] . The link between glycan structures and cancer has received particular attention, and glycobiology has become a new tool for cancer therapeutics and diagnostics. Some examples of biosensors devoted to detecting carbohydrate-relevant species have been reported in literature [59] , in many cases coupled to nanomaterials [60] . Hybridization-based biosensors

In the last decades, NA-based biosensors have gained much interest in various clinical applications. An NA biosensor is defined as an analytical device incorporating an oligonucleotide, even a modified one, with a known sequence of bases, or a complex structure of NA (like DNA from calf thymus) either integrated within or intimately associated with a signal transducer [61] . NA biosensors can be used to detect DNA/ RNA fragments or either biological or chemical species. Most NA biosensors are based on the highly specific hybridization of complementary strands of DNA or RNA molecules; this kind of biosensor is also called a genosensor [62] . The probe, immobilized onto the transducer surface, acts as the biorecognition molecule and recognizes the target DNA/RNA, while the transducer is the component that converts the biorecognition event into a measurable signal. Assembly of numerous (up to a few thousands) DNA biosensors onto the same detection platform results in DNA microarrays (or DNA chips), devices, which are increasingly used in clinical diagnostic. In NA biosensors, the detection of the hybridization event has been carried out through different detection technologies, from label free methods, such as piezoelectric and surface plasmon resonance (SPR) transduction, to other methods often requiring labels, such as electrochemical techniques. Several reviews have recently appeared in the literature [16,63–66] elucidating all the critical aspects related to the transduction step. As the specificity of the hybridization reaction is essentially dependent on the biorecognition properties of the capture oligonucleotide, design of the capture probe is undoubtedly the most important pre-analytical step. The probes can be linear oligonucleotides or structured (hairpin) oligonucleotides, which are being used with increasing frequency [65,67] . Peptide NAs (PNAs) and locked NAs (LNAs) are frequently used in DNA biosensor to develop the capture probe sequence [68] . PNAs are DNA mimics in which the nucleobases are attached to a neutral


3425

Review Palchetti

A

B Head

Tail

Figure 2. Some examples of bacteriophages. (A) Transmission electron microscopy image of a Multiple M13 bacteriophages and (B) schematic of an icosahedral bacteriophage drawn by ChemDraw. Reprinted with permission from [53] © John Wiley & Sons (2009).

N-(2-aminoethyl)-glycine pseudopeptide backbone, whereas LNA is an NA analog of RNA, in which the furanose ring of the ribose sugar is chemically locked by the introduction of a methylene linkage between 2′-oxygen and 4′-carbon. In addition to specificity, sensitivity is also a key factor in the performances of a biosensor: sensitive detection of specific gene sequence on the basis of the hybridization reaction can be achieved by increasing the probe immobilization amount and controlling over the molecular orientation of the probes. Nanomaterials have been increasingly introduced in the fabrication of biosensors for this purpose, in order to increase the immobilization amount of the DNA probe and to magnify the detection signal and lower the detection limit. The use of a label may also help to improve sensitivity. The experimental variables affecting the hybridization event at the transducer–solution interface are referred to as stringency and they generally include hybridization and posthybridization washing buffer composition and reaction temperature. In recent years, different genosensor formats have been applied to a multitude of genetic diseases including cancer. An emergent challenging area in clinical diagnostics is that of miRNA detection. Some examples of genosensors for miRNA analysis are reported in the following section. An emerging application: miRNA determination

miRNAs (miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) [69] that act as regulators of protein translation. Because many diseases are caused by the misregulated activity of proteins, miRNAs have also been implicated in a number of diseases including a broad range of cancers [70] . Consequently, miRNAs are intensely studied as candidates for diagnostic and prognostic biomarkers. Moreover, the ability to selectively regulate protein activity

3426


through miRNAs could play a role in the treatment of many forms of cancer and other serious illness [70] . miRNAs have a peculiar biogenesis [71] (Figure 3) . Initially, miRNAs are part of a larger piece of RNA (primary-miRNA or pri-miRNA or primiR) containing stem-loop structures that may contain multiple potential miRNAs. Pri-miRNAs are transcribed by RNA polymerase II or III, which are subsequently processed in the nucleus by the RNase III endonuclease Drosha and DGCR8 (the ‘microprocessor complex’) to form intermediate stem–loop structures approximately 70 nucleotides long called ‘precursor miRNAs’ (pre-miRNAs or pre-miRs). These pre-miRNAs fold to form imperfect stem–loop structures that are transported with the help of Exportin-5 from the nucleus to the cytoplasm, where they undergo further processing by another RNase III endonuclease, Dicer. Dicer removes the loop of the pre-miRNA to produce an imperfect duplex (miR:miR*) made up of the mature miRNA (miR) sequence and a fragment of similar size (miRNA* or miR*), which is derived from the opposing arm of the pre-miRNA. The miRNA strand of the duplex is loaded onto the RNA-induced silencing complex (RISC); the miRNA* separates from the duplex and is degraded [70] . There are several methods for detecting miRNAs, mostly relying on hybridization [72] . The most standardized and widely used method to detect miRNAs is northern blotting. In this method, the sample containing miRNAs is run on an electrophoresis gel. Then, the miRNAs are transferred to a nitrocellulose membrane, followed by soaking in a solution containing a fluorescent or radiolabeled oligonucleotide probe which is complementary to the target miRNA for hybridization to occur. After nonhybridized DNA has been removed, the miRNA target can be detected. Unfortunately, the low sensitivity, the time consuming and the laborious procedures make northern blotting difficult for routine miRNA analysis in a clinical setting. Moreover, northern blotting is not considered high throughput. Currently, the most widely used high-throughput method to detect miRNAs is through microarray. Microarrays permit the simultaneous detection of hundreds of miRNAs per time. A set of oligonucleotide capture probes are spotted on glass slides, and a sample of extracted RNA enriched for small molecule RNAs is allowed to hybridize with the capture probes. The most important disadvantage of microarray technologies resides in the nonquantitative nature of this method, which, therefore, requires further experimental validation [70] . Quantitative real-time PCR is becoming a popular method to detect miRNAs due to its high level of sensitivity (only a few picograms of miRNAs are required), accuracy and ease-of-use. In general, the target miRNA



is first reverse transcribed into its cDNA, which will subsequently be amplified via PCR. However, the simple translation in PCR is obscured by the small length of mature miRNAs that share the same length as normal PCR primers. Shorter primers are typically not useful, as they demand very low melting temperature that eventually affects the efficiency of PCR. Although several primer extension techniques have been suggested to combat the intrinsic shortcoming of mature miRNAs, a great deal of effort has been devoted to developing analytical methods for miRNA analysis that possess appropriate sensitivity, appropriate dynamic range and multiplexing capability without PCR. At this purpose, different biosensor platforms have been proposed [72–75] . Electrochemistry is an emerging technique for miRNA biosensing [76–78] . A search on the term ‘miRNA biosensor’ and a refining of the results for the term ‘electrochemical,’ using SciFinder®,

Review

revealed that papers focused on electrochemical biosensors are around 40% of all the papers on miRNA biosensors. Electrochemical detection of miRNA was first reported by Gao and Yang in 2006 [79] using electrocatalytic nanoparticle tags. The assay was based on a direct chemical ligation procedure involving a chemical reaction to tag. The nanoparticles catalyze the oxidation of hydrazine and enhance the detectability of miRNAs, thereby lowering the detection limit to femtomolar level. Labeling of miRNAs for electrochemical assay is often necessary for miRNA sensitive detection. Gao and Yu reported a miRNA labeling procedure that utilizes a chemical ligation to directly label miRNA, via a covalent bond, to an isoniazid-substituted osmium complex [80] . In a separate attempt, the same group made use of the Ru(PD)2 Cl2 (PD = 1, 10- phenanthroline-5,6-dione) electrocatalytic moiety to monitor the oxidation of

Nucleus Cytoplasm

Pol II Pri-miR 5´

3´

DGCR8 Drosha Exportin 5 3´

5´

5´ Helicase 3´

3´

Dicer Translational repression

5´

RISC Mature miR strand incorporated into RISC

mGpppG

AAA...An 3ÚTR

7

mRNA cleavage

RISC

RISC mGpppG

AAA...An 3ÚTR

7

RISC-independent functions Decoy activity

Translational activation RISC 5ÚTR

Figure 3. Biogenesis and mechanisms of action of miRNAs: an overview. Reprinted with permission from [70] © John Wiley & Sons (2012).



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Review Palchetti

Table 4. Some examples of recent electrochemical miRNA biosensing platforms in label-free format. Transducing principles

miRNA

Limit of detection

Ref.

Square wave voltammetry

miR-141, miR-103, and miR-29b-1

650 fM

[134]

Electrochemical impedance spectroscopy

miR-let-7 a, miR-let-7 b, miR-let-7 c

1.0 fM

[135]

Cyclic voltammetry

miR-155

1.87 pM

[136]


miR-let-7 a, miR-let-7 b, miR-let-7 c

2.0 fM

[137]

Differential pulse voltammetry

mir-21

45 fM

[138]


miR-720, miR-let-7c, miR-1248

2.0 fM

[139]


miR-24

1 pM

[140]


miRNA-16, miRNA-15a and miRNA-660 4.3 pmole in 3 ml samples

[141]

Amperometry

miR- let7a

[142]

13.6 aM

hydrazine [81] . Both these biosensors were applied to the quantitation of miRNA in total RNA extracted from HeLa cells. Recently, Gao group [82] reported the use of ruthenium oxide (RuO2) nanoparticles initiated polymerization of 3,3′-dimethoxybenzidine and miRNA-templated deposition of an insulating poly(3,3′–dimethoxybenzidine) film. In a separate work by Peng, RuO2 nanoparticles tagged to target miRNA strands served as a catalyst for polymerization of aniline and the hybridized miRNA strands acted as templates for the deposition of PAn at the hybridized miRNA strands [83] . Enzymes have been used as label in many biosensor formats. Pohlmann et al. [84] used an esterase as amplifier in a gap-hybridization assay format. Another strategy for miRNA detection is through the use of an

electrochemically activated glucose oxidase (GOx) tag/ amplifer [85,86] . Yin et al. [87] reported a miRNA biosensor based on triple-signal amplification due to the immobilized graphene and dendritic gold nanostructure and subsequent monitoring of the reduction current as a result of the oxidation of hydroquinone by H2O2 and horseradish peroxidase. Bettazzi et al. reported an electrochemical method based on paramagnetic beads and alkaline phosphatase amplification [88] . Yang and colleagues [89] developed a biosensor based on palladium (Pd) nanostructured microelectrodes. Differential pulse voltammetry (DPV) was used to monitor the electrochemical reduction current of Ru3+ accumulated on the electrode surface after hybridization with the target miRNA. The signals observed with this reporter system (Ru3+) are amplified by the inclusion of ferricyanide, which can regenerate Ru3+

Table 5. Some examples of electrochemical miRNA biosensing platforms in label-based format. Transducing principles

Label

miRNA

LOD

Ref.


DNAzyme

miR-21

0.006 pM

[143]


Protein 19

miR-21

–

[144]


DNA concatamers

miR-21

100 aM

[145]


–

miR-let-7 a, miRlet-7 b, miR-let-7 c, miR122

98.9 fM

[146]

Differential pulse voltammetry and chronoAmperometry

MB - ALP

miR-222

7 pM

[88]

Square wave voltammetry and P19 protein electrochemical impedence spectroscopy

miR-21, miR-32, and miR-122

5 aM

[92]

Amperometry

HRP

–

1 fM

[147]

Amperometry

HRP

miR-122

10 aM

[148]


MB - Os(VI) complex

miR-522, miR-261 10 nM

[149]

Amperometry

AuNPs-ALP- p-AP

miR 21

[150]

3 fM

ALP: Alkaline phosphatase; AuNPs: Gold nanoparticles; HRP: Horseradish peroxidase; MB: Magnetic beads; pAP: Para aminophenol.

3428




chemically after its electrochemical reduction. After a 30 min hybridization, the detection of 10 aM of target was reported and the RNA extracted in a panel of RNA samples tested. Label-free detection schemes have been also reported. In 2007, Fan and colleagues [90] introduced a strategy for sensitive and label-free detection of miRNA based on conducting polymer nanowires. Zhang and colleagues [91] proposed a biosensor based on PNA capture probes immobilized on silicon nanowire. Resistance change measured before and after hybridization correlates directly to concentration of the hybridized target miRNA. A detection limit of 1 fM was obtained. Labib et al. [92] introduced a three-mode electrochemical biosensor that exploited the strong and nonsequence-specific binding of a p19 fused dimer protein to double-stranded miRNAs and incorporated three modalities based on hybridization, p19 protein binding and protein displacement. Some examples of electrochemical biosensor based assays, recently reported in literature, can be found in Tables 4 & 5. Field effect, photoelectrochemical and electrochemiluminescent phenomena will be not reviewed here, since we focused only on classical electrochemical transduction principle, even if these techniques have been applied for miRNA determination. Examples of label-free detection are reported in Table 4, while several label-based approaches are reported in Table 5. The works reviewed show that miRNA diagnostics is becoming an increasingly relevant field of application of biosensor technology. Nevertheless, most of the current biosensor-based assays show a lack of reliable validation in clinical samples. Current challenges of affinity biosensors for cancer-related diagnostics One of the main challenges for biosensors is their implementation as point of care (POC) tools. POC systems are viewed as integrated systems that can process clinical samples for a number of different types of biomarkers in a variety of settings, such as clinical laboratories, operating theaters, doctor’s offices and eventually at home, providing opportunities for rapid screening of at-risk patients, surveillance of disease recurrence and better monitoring of treatment. Miniaturization of biosensor devices is of fundamental importance to reach this goal. Miniaturization allows for a large number of analyses to be made, also in a multiplexed format (i.e., analyses of different analytes simultaneously), using less reagents, time and effort. Most biological assays require labor-intensive and time-consuming processes, including incubation steps, washing steps, preconcentration steps, addi-


Review

tional reagents, which can be time consuming. The integration of biosensors in microfluidic platforms helps the miniaturization goal, reducing the assay time and sample and/or reagent consumption as well and serving automation purpose [93] . This fact is really important in order to reach widespread pointof-care use. Another important issue that can be easily covered by microfluidics is the possibility of multiplexed analysis [94–97] . An interesting electrochemical approach is that reported by Maholtra et al. [96] for the simultaneous detection of IL-6, IL-8, VEGF and VEGF-C [96] in human serum, with detection limits in the range of 5−50 fg/ml. Magnetic transducers have also been applied to the sensitive detection of different cancer biomarkers in a diverse range of biological fluids [98] in a microfluidic setting. However, significant challenges have to be overcome before biosensor-based point-of-care devices for cancer diagnosis become a reality [94,99] . Accuracy is, obviously, together with sensitivity, selectivity and precision, an important parameter that should be carefully controlled through validation processes, before a new device will find application. If a multitude of intriguing detection schemes has been reported in literature, also for in vivo diagnostics, it should be noted that extensive validation in clinical samples is not always reported. Surely, before application of any assay, including biosensors, in cancer diagnosis, prognosis and theragnosis, there is the need to ensure robust technical and clinical validation of the developed bioassays and biosensors and preferably within the context of randomized clinical trials in which the detection of particular molecular biomarkers by means of a particular methodology can be clearly linked to patient outcome. Conclusion Thousands of biosensor-based assays for the detection of cancer biomarkers have been reported in literature so far, highlighting the importance of biosensor technology in this field. The increasing number of new biological derived semisynthetic or synthetic receptors is allowing the development of more robust, versatile and stable biosensors, while nanotechnologies are facilitating highly sensitive and innovative transduction of the binding event. Our concluding remark regards commercialization of biosensors and their application to practical clinical analysis. Commercialization of biosensors has not yet maintained pace with the large amount of research activity. Several are the reasons of this phenomenon [8,9] . Both cost and technical issues are


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Review Palchetti slowing down the commercialization of new devices. However, it is important to continue working on biosensor technologies, to allow a successful transition from the research laboratory to clinical settings. Future perspective The literature reviewed in this paper demonstrates that biosensors are capable of characterizing and quantifying cancer-related biomolecules and even cancer cells. The identification of candidate cancer drugs and candidate cancer biomarkers [100] are other scenarios that will be benefit from biosensor features and from the discovery of innovative biorecognition elements [101–104] . Recently, aptamers, evolved from improved SELEx process, have been proposed for multiplexed proteomic technology for novel biomarker discovery. Furthermore, aptamers, engineered proteins and other nature inspired molecules are themselves candidates as cancer drugs or for drug release strategies. The coupling of these molecules with new nanomaterials and nanocomposites will offer exciting possibili-

ties for hybrid devices that combine both diagnostic and therapeutic components and will be important for in vivo diagnostics and for in vivo monitoring of therapy outcomes. As already stated, work remains to be done before biosensors can be used in cancer clinical practice. Miniaturization will still be an important issue in this sense. However, the future is bright for producing microfluidic, multiplexed tools, within the next decades, which can be used as point-of-care cancer diagnosis and therapy. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary • Affinity biosensors might be powerful tools for cancer diagnosis, prognosis and theragnosis. • Biological, semisynthetic or synthetic bioreceptors with different affinities, specificity and chemical composition toward cancer biomarkers are nowadays available. • Antibodies still play an important role for the detection of proteins, like prostate-specific antigen or carcinoembryonic antigen, hormones and other cancer-related molecules. • Aptamers, engineered proteins, peptides and molecularly imprinted polymers are promising candidates in the development of synthetic or semisynthetic ligands that can deliver the sensitivity and selectivity of biological systems with an increased stability. • Glycans/carbohydrates are emerging new receptors to study glycan expression profiles in cancer cells. • Nucleic acid biosensors are especially used for the detection of marker genes. A challenging field of applications of nucleic acid based biosensors is microRNA determination. • A current challenge for biosensor technology is the implementation as point of care tool. Microfluidics can help to reach this goal. 5

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Description of different bioassays for the analysis of protein biomarker with potential for multiplexed point-of-care applications.


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ferrocene functionalized iron oxide nanoparticles. Biosens. Bioelectron. 26(8), 3590–3595 (2011). 124 Liu J, Zhou H, Xu JJ, Chen HY. Dual-biomarker-based

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Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

Yeast dual-affinity biobricks: Progress towards renewable whole-cell biosensors.

A mathematical method for extracting cell secretion rate from affinity biosensors continuously monitoring cell activity.

Biosensors and flow injection analysis.

Biosensors in forensic analysis. A review.

Analysis of mathematical modelling on potentiometric biosensors.

Biosensors for process control.

Biosensors for environmental monitoring.

Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor.

Biosensors for Monitoring Airborne Pathogens.

Biosensors.

Surface plasmon resonance (SPR) biosensors in pharmaceutical analysis.

Chemically modified diamondoids as biosensors for DNA.

Fish scales as biosensors for catecholamines.

Biosensors for hepatitis B virus detection.

Biosensors for the Detection of Food Pathogens.

Nanomaterial-based biosensors for food toxin detection.

Aptamer-based biosensors for biomedical diagnostics.

Biosensors. NO release for good measure.

FET biosensors.

Calorimetric biosensors.

Versatile matrix for constructing enzyme-based biosensors.

Tyrosinase-Based Biosensors for Selective Dopamine Detection.

Amperometric biosensors.