Sensors 2008, 8, 1366-1399

sensors
ISSN 1424-8220 © 2008 by MDPI www.mdpi.org/sensors Review

Assembling Amperometric Biosensors for Clinical Diagnostics
María Soledad Belluzo, María Élida Ribone and Claudia Marina Lagier * Analytical Chemistry Department, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario –2000- Argentina. * Author to whom correspondence should be addressed. E-mail: clagier@fbioyf.unr.edu.ar Received: 1 February 2008 / Accepted: 14 February 2008 / Published: 27 February 2008

Abstract: Clinical diagnosis and disease prevention routinely require the assessment of species determined by chemical analysis. Biosensor technology offers several benefits over conventional diagnostic analysis. They include simplicity of use, specificity for the target analyte, speed to arise to a result, capability for continuous monitoring and multiplexing, together with the potentiality of coupling to low-cost, portable instrumentation. This work focuses on the basic lines of decisions when designing electron-transfer-based biosensors for clinical analysis, with emphasis on the strategies currently used to improve the device performance, the present status of amperometric electrodes for biomedicine, and the trends and challenges envisaged for the near future. Keywords: Biosensor, electrochemical, amperometric, clinical diagnosis

1. Introduction Current methodologies to determine the species that define or identify one particular clinical condition present several drawbacks, even when leading to reliable results. Requirements of previous separative steps, the need to count with highly trained personnel to perform the analysis, its frequent high cost, the circumscription to state-of-the-art laboratories are, among others, the main reasons that have prompt the search for new analytical technology to achieve advantageous clinical diagnostic methods [1]. Biosensors emerge as upbeat technology to face this challenge. Biosensors have been defined as compact analytical devices that bring together the use of a biological, a biologically-derived or a biomimic element to recognize the analyte. They are closely associated with, or integrated to, a signal conversion unit, the so-called physicochemical transducer,

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which eventually leads to a read-out. The use of biological materials as recognizing elements gives biosensors a remarkable ability to specifically react with the analyte of interest, distinguishing it from structurally similar compounds. Taking into account the biomolecule that recognizes the target analyte, biosensors can be named as (i) affinity sensors, when the bioreceptor uses non-covalent interactions like antibody-antigen reactions or DNA strand hybridisation, and (ii) catalytic or enzyme sensors, when the analyte is the enzyme substrate, or it can be detected by measuring the signal produced by one substrate or product of the enzymatic reaction involving the analyte. Biosensors are also classified according to the parameter that is measured by the physicochemical transducer of the biological event. Thus, classically biosensors are grouped into optical, electrochemical, acoustic and thermal ones. Considering that electrochemical reactions directly generate an electronic signal, biosensors based on this approach greatly simplifies signal transduction, avoiding expensive equipment requirement. Among electrochemical biosensors, the oldest ones, which have led to the higher number of readyto-use devices, are based on the monitoring of electron-transfer processes, thus belonging to the amperometric category. This review will centre on this group. The signal of these biosensors is generated by the electron exchange between the biological system in the bioreceptor layer and one electrode. Generally speaking, when using amperometric biosensors, the analyte undergoes, or is involved, in a redox reaction that can be followed by measuring the current in an electrochemical cell. The analyte, or the species involved with it via a (bio)chemical reaction, changes its oxidation state at one electrode. The electron flux is then monitored and is proportional to the amount of the species electrochemically transformed at the electrode. Figure 1 depicts the working principles of an amperometric biosensor. Figure 1. Scheme depicting the functional principles of an amperometric biosensor.
= analyte = bioreceptor

esignal = current transductor sample applied potential

The typical electrochemical technique performed when working with electron-transfer-based biosensors is chronoamperometry, in which the current is measured as a function of time, when the electrode is driven at an appropriate constant potential. Figure 2A shows a standard curve obtained when using an amperometric bioelectrode, in which the current varies upon the addition of a particular compound (e.g. a redox-enzyme substrate) to render a particular product that is electro-transformed at the electrode. The current change registered is proportional to the amount of electro-oxidized/reduced species, which in turn may be directly or inversely proportional to the analyte concentration, depending on the assay format.

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Other electrochemical techniques, based on the electrochemical oxidation/reduction of species directly or indirectly involved in the recognizing biological reaction have been used when dealing with electron-transfer based biosensors. For example, it is possible to follow the amount of a species that is electro-transformed at one electrode by monitoring the time taken to perform such a reaction, when working at constant current, and varying the electrode potential. Fig. 2B depicts a typical curve obtained when carrying out one of these experiments, named potentiometric stripping analysis. The peak area is proportional to the time necessary to electro-transform a species attached to the electrode, which, for example, may be directly proportional to the analyte concentration. Figure 2. Typical plots of the signals obtained when using electrochemical biosensors based on the amount of electro-transformed species at the electrode by means of (A) chronoamperometry, and (B) potentiometric stripping analysis.

time (s)

dt / dE (s V-1)

i (μA)

potential (V)

A

B

Several other electrochemical techniques are also employed when using electron-transfer-based biosensors, such as stripping voltamperometry, differential pulse voltamperometry, coulometry, etc. These methodologies essentially measure parameters related to the amount of electro-transformed material on the electrode as current flows in the cell. Clark and Lyons presented the precursor of today’s biosensors in the early sixties [2], though it was named “the enzyme electrode” rather than a glucose amperometric bioelectrode (Figure 3). The analyte, glucose, diffuses through a dialysis membrane to reach the trapped enzyme, glucose oxidase, which is placed very close to the surface of a platinum electrode. In the presence of the enzyme and oxygen, the analyte produces glucuronic acid and hydrogen peroxide. Monitoring the decrease in oxygen concentration allows to determine the glucose concentration of the medium bathing the outside face of the dialysis membrane. In this case, oxygen is reduced at the metal electrode polarized with a constant potential, and the current flowing in the cell is proportional to the oxygen concentration in the solution. Soon after Clark’s proposal, Updike et al. introduced modifications to this first approach to avoid oxygen concentration dependence. They monitored the current flowing in the cell when one of the products of the enzymatic reaction, hydrogen peroxide, was oxidized on an electrode at a convenient potential [3]. Today, Clark’s brilliant, pioneering development has evolved, reaching the markets as “bloodglucose, self-testing biosensors”. It is routinely used for screening and treating diabetes -an illness

4% of the population in industrialized countries. Figure 3.Sensors 2008. Figure 4 summarizes the different bioreceptors we can use to achieve the desired selectivity. SelectivitySpecificity . thus conferring speed to the analysis. It should be noticed that this is the foremost feature of biosensors. 8 1369 whose prevalence is ca. In clinical-chemical analysis. any biological or biologically derived element can be used. we deal with biological samples.2. This choice depends on the analyte properties and structure. which display a complex matrix likely carrying interferences. since provides the devices the capacity of working without previous treatments of the sample. Selectivity-Specificity 2.0. in principle. Illustration of Clark’s enzyme electrode displaying the reactions occurring at the inner solution containing the enzyme and at the electrode surface. .1. Choosing the bioreceptor system The bioreceptor layer is responsible for the selectivity/specificity of the device and. according to the nature of the analyte.the equipment being worldwide sold by leader sensor companies [4].6 V Ag/AgCl half saturated KCl teflon membrane dialysis membrane oxygen glucose enzyme (Ez) Pt [O2] glucose + O2 Ez glucuronic acid + H2O2 2. and on the presence of similar interfering substances potentially present in the sample. The first task when designing a biosensor is to select a suitable bioreceptor system so that the recognition sites interact specifically with the target analyte.

In principle. we can use redox enzymes that react with the analyte producing products that can be monitored by amperometry via their oxidation/reduction on an electrode. an important issue is regaining the original oxidation state of the redox active site. which involves an enzyme that changes the oxidation number of the . 2. it is not feasible to oxidise/reduce the active site of one enzyme by setting directly the electrode potential at any particular value (an exception will be discussed later regarding oriented attachment of bioactive materials in point 3. so that the active site is electrically insulated. Though it would be desirable to electrochemically detect changes in the enzyme substrate (the analyte) or in one reaction product. which transport the charge between the enzyme active site and the electrode surface. Most of the enzymes used in biosensor technology have their redox centre inside a pocket or at the core of the protein.Sensors 2008. This difficulty is commonly tackled by using redox charge mediators. most of the analytes of clinical interest are not natural substrates of a redox enzyme. They are based upon the fact that it is feasible to transform a nonredox reaction into a redox one.2. 8 1370 Figure 4. depending on the analyte chemical properties. when designing enzyme-based biosensors. Enzymes The extraordinary specificity displayed by enzymes for their natural substrates makes them the first candidates to be incorporated as the biorecognition element. Generally speaking. It is worth mentioning here that. as it was illustrated above with Clark’s glucose biosensor. so as to keep the enzyme active to react further with its substrate. This subject will be discussed later in section 2.1.1). Bioreceptors eligible for attachment to the electrode surface. A variety of strategies have been developed to work this problem out.1.

which bind to such Agn with remarkable specificity. In this case. when the clinical analyte of interest is an Agn. it has been proposed the use of coupled enzymatic reactions following the one where the analyte participates. Figure 5A depicts the competitive immunosensor format. allows to perform a competitive assay. enters one individual’s body. Abs usually interact exclusively with their complementary Agns. Antibodies. 2. Consequently.Sensors 2008. They include amperometric biosensors to determine urea [6]. antigens & aptamers When a foreign compound. Abs can be either produced by inoculation of the Agn to an immuno-competent individual.1. its immunological system generally produces proteins. 8 1371 species that can be monitored by amperometry. to detect creatine. and creatinine. thus conferring a remarkable specificity to the device. since decades. creatinine amidohydrolase creatinine + H2O creatinine amidinohydrolase creatine (1) creatine + H2O sarcosine oxidase sarcosine + urea (2) sarcosine + H2O + O2 glycine + formaldehyde + H2O2 electroactive species (3) Other biosensors to determine analytes of clinic relevance have been developed. added together with the sample carrying the analyte. the species that is electrochemically transformed at the electrode to generate an amperometric signal.2. using redox enzymes that allow for the detection of the oxidation or reduction of one product directly or indirectly involved in the enzymatic reaction. a myriad of research work aimed to clinical-chemistry analysis was carried out [13]. one indicator of kidney function [5]. The coupled reactions are shown in equations 1 to 3. an important product of protein catabolism. The design below has been proposed by Tsuchida et al. or it can be chemically modified to behave as an Agn. They ultimately produce hydrogen peroxide. it is possible to produce Abs against almost any molecule (analyte). currently. lactate [7-9] and pyruvate [10]. even those unable to stimulate naturally the immune system [12]. Thus. Nowadays. Moreover. antibodies (Abs). enzyme linked immunosorbent assays (ELISA). These enzymes are sometimes used as label or reporter of the biological recognition event. or by genetic engineering of bacterial or animal cells [11]. in a similar way to competitive. where the target analyte competes with the labelled antigen for the biorecognition sites of the antibody attached to the . it is possible to use the complementary Abs as capturing molecules in the bioreceptor system. Biosensor technology has taken advantage of the high specificity of the Agn-Ab reaction and. the biosensor will be called immunosensor. named after the usage of Abs as bioreceptors. among others. called antigen (Agn). to finally produce a species detectable by amperometry. The use of one enzyme-labelled antigen.

the most serious parasitosis in South America. the signal diminishes as the analyte concentration increases. a pregnancy-indicator hormone. enzyme-labelled secondary Ab towards a particular region of the analyte (e. creatinine [20]. such as prostate specific antigen [14. False-positive results are generally associated with the occurrence of non-specific Abs. human chorionic gonadotrophine (hCG) [17]. have been detected with this strategy. Scheme depicting different immunoassay formats using amperometric detection. Figure 5B illustrates the working principle of an indirect immunoassay format with amperometric detection. sometimes.Sensors 2008. the bioreceptor is the Agn. which is transformed at the electrode. Figure 5. when a patient is infected with a . In this case. which captures the specific Ab from the sample. progesterone [18]. the hormone indicative of ovary function. After rinsing the system. or reacts with one electroactive enzyme co-substrate further added. and by Ferreira et al [25]. For instance. cross-reactions may occur. (A) Biosensor to detect an antigen using a competitive immunoassay format. one expects the signal to increase with the analyte concentration. among others.analyte target analyte Substrate Ez-labelled secondary Ab target analyte Substrate Ez Product Ez Product ecapturing Ag Electrode capturing Ab Electrode e- A B Diagnostic electron-transfer-based immunosensors have been reported to detect cancer markers.27] and hantavirus [28].23]. with a redox-enzyme-labelled antigen and the natural substrate of the enzyme. The reader is referred to wide-ranging immunosensor reviews such as those listed in references [13. cholesterol [19]. the addition of the redox-enzyme-label substrate generates the product. and auto-antibodies of Type I diabetes [21]. 8 1372 electrode. similar to that used to detect Agns.15] and carcinoembryonic antigen [16]. the Fc region of human immunoglobulin G. to detect Chagas’ disease. leading to a detectable current. such as those caused by Schistosoma japonicum [26. Biosensors using this format have been proposed by our group [24].g.22. In this case. which are often developed by individuals suffering from other diseases. Despite the outstanding specificity of the Agn-Ab reaction. see below) will be captured on the surface of the modified electrode. labelled. When the analyte is an Ab. which is usually the case when diagnosing an infection. another immunological assay scheme can be performed. IgG. Other clinically relevant infections. In this competitive assay. A subsequently added redox. Ez. The occurrence of a current once the substrate of the enzyme label is added evidences the presence of the analyte in the sample. (B) Biosensor to detect a specific antibody using an indirect immunoassay format.

42. which wrinkle the oligonucleotide chain. as well as others to determine thrombin [46-48].52]. where each nucleotide base strongly binds to its complementary . Moreover. Another related aptasensor is the one used to determine simultaneously lysozyme and thrombin [50]. etc. Nucleic acids Clinical-chemistry analysis often involves the detection of specific nucleotide sequences related to either a particular microorganism. for example. this individual may have produced Abs against orthologous proteins of both infective agents. one to detect 17β-estradiol. lipids. Results obtained with amperometric biosensors assembled with the new synthetic proteins have been presented in a preliminary work [31]. a hormone indicative of ovary function [44].3.34]. but also to simplify their attachment to the electrode surface (see below. serum lysozyme has been proposed to be a marker of chronic inflammatory diseases. which varies with the nucleotide sequence. 8 1373 microorganism phylogenetically related with the one whose infection is to be determined. aptamers have also been used as analyte-capturing species. antibodies. do not lose their remarkable specificity [35. aptamers present a three-dimensional shape that confers the molecules the aptitude to interact either strongly or weakly with partner molecules. electron-transferred based aptasensors to determine small molecules like cocaine in biological fluids have been also developed [45]. Extensive and detailed aptamer-based sensor technology is reviewed in references [33.37-43]. scientists have preferred the use of aptamers as recognizing element when developing biosensors [33.2. Our group has synthesized such proteins for chagasic infection diagnosis. item 3.36]. or DNA mutations that originate genetic diseases. in the lately years.to 40-nucleotide length. as well as of leukemia [51]. proteins. such as sarcoidosis. The enormous diversity of aptamer conformational structure. In these cases. The use of recombinant DNA machinery to obtain specific.Sensors 2008.g. The recombinant proteins produced allowed not only to obtain a unique analyte-capturing bioreceptor. synthetic recombinant proteins can assist to work the problem out. Likewise. depending on their structure. but also to attach it to the electrode in an oriented manner.1. triggered by highly specific base-pairing interactions. Therefore. at variable settings. They allow not only to obtain the analyteexclusive bioreceptor. thrombin is the main executioner of the coagulation cascade. tailor-made proteins. tuberculosis and liver and bowel inflammatory diseases. which may be the causal agent of an infection. Aptamers are linear nucleotide sequences. or that are connected with high probability to develop certain type of diseases.33]. These are the reasons why. Therefore. DNA. Recently. carbohydrates. the use of natural proteins of the microorganism as capturing Agn may lead to incorrect results. [33. aptamers are easily synthesised and isolated. undergoing many intramolecular interactions.). Thus. together with biosensor technology. as for example.34. emerges as an auspicious means to achieve ready-to-use devices. Nucleic acid hybridisation is a thermodynamically favoured process. e. Numerous clinically related aptamer-based biosensors have been designed. allowing to build up robust biosensors that. Recombinant proteins are generally synthesized from DNA sequences engineered to encode peptide fragments where the specific regions responsible of cross-reactivity have been excised [29. 2.30]. allows them to be selected according to the strength of interaction with their partner molecule [32. They are more stable than. usually of 15. and its determination in blood is useful to assess the haemostatic status of patients for the diagnosis of coagulation and thrombotic disorders [49]. including among them virtually any kind of analyte. aptamers can be chosen so as to tightly and selectively bind to target molecules.

mostly by using an electrochemical reporter of the hybridisation [53-56]. can be oxidised at a particular potential. an hypoxantine-derivative nucleoside. firstly rendering 7. A variety of other intercalating compounds. The central metal ion can be electrochemically transformed when is close to the electrode surface. Once the binding partners have met.64]. 8 1374 base through several hydrogen bonds. In the minimal configuration of amperometric DNA. and have proposed a number of strategies to detect hybridisation [60-62]. the purine base guanine (G). making this a suitable platform to detect the event.or RNA-based biosensors. To overcome this inconvenience. and the presence of pathogenic Escherischia coli bacteria [53]. the event is detected using different approaches. nucleic acid sequence is anchored to an electrode to capture the analyte. Another similar compound. The two latter ones have been applied to detect the gene encoding for apolipoprotein E. This compound was used to detect the three-base deletion of ∆F508 DNA sequence.10-phenanthroline)33+. some of them included in references [63-68]. since they are comprehensive reviews dealing with DNA-based biosensors. For example. or to steric hindrance that prevents accessibility of the oxidation sites [80]. was also used to detect DNA sequence of Mycobacterium tuberculosis [82].85]. Cryptosporidium parvum [83]. such as methylen blue [86]. Co(1. and references therein]. indicators of chronic myelogenous leukemia [71].2'-bipyridy1)33+. The simplest strategy to detect nucleic acid hybridisation relies on monitoring the direct oxidation/reduction of nucleotide bases. Accordingly. daunomycin and aromatic amines [87]. The use of cationic metal complexes that can associate to double-stranded (ds-) DNA stronger than to ss-DNA has been also proposed [53. Clinical applications using DNA-amperometric-based biosensors include devices potentially useful to determine indicators of pathogenic bacteria [69]. and acute promyelocytic leukemia [72]. have been used to evidence DNA hybridisation. A vast literature has been published on DNA hybridisation detection. proved to be useful [56. In this case. The most important drawback that presents this simple method to determine hybridisation is the high background current in the absence of the hybridisation event [75]. since its preferential base partner is the same as for G [81]. there is a signal increase after pairing of DNA complementary strands. virus DNA sequence such as that of SARS virus [70]. and therefore the signal increases as long as hybridisation takes place. Therefore.54]. In principle.Sensors 2008. small complexes can intercalate between DNA grooves possessing a much higher affinity for the DNA hybridised compared to ss-DNA. the replace of G base by inosine. DNA/RNA strands are excellent applicants to be used as recognizing elements to build up bioreceptor layers. Since the early sixties. Palecek and co-workers have been studying nucleic acids electrochemically [57-59]. hepatitis B virus [84]. a single-stranded (ss-).8-dihydro-8-oxoG (commonly referred to as 8-oxoG). a protein involved in familial dysbetalipoproteinemia [87]. this has been ascribed to the molecule rigidity gained after hybridisation [79. the number of G residues present in the ss. Still another strategy taking advantage of labelled-complementary base sequences has proved to be effective to monitor .nucleic acid probe will lead to a directly proportional reduction or oxidation signal. We refer readers to references [73-75]. in this case. which suffers further oxidation reactions [76-78] (Figure 6A). its complementary DNA or RNA strand. an indicator useful to diagnose cystic fibrosis [54]. It should be considered that the G electrochemical signal diminishes upon base pairing. efficiently sticking the bases together. and the human immunodeficiency virus [82. A widely used DNA-intercalating metallic complex is Co(2.

1.g.2. interferences may also be electrochemically oxidised/reduced. . since other compounds present in the sample matrix may also be transformed on the electrode set at the chosen potential. For example. For a deeper insight on the subject. acetaminophene. Avoiding interfering reactions Though there are many enzymes whose products can be monitored by amperometry. we refer the readers to exhaustive reviews. 3. 2. namely: (i) preventing the interfering compound from occurring in a significant amount nearby the electrode and (ii) working at potentials at which oxidation/reduction of interferences is negligible. and is afterwards electro-transformed. which bounds to the nucleic acid probe. cysteine. The same holds true when directly detecting nucleic acid hybridisation via oxidation of G. The signal increases upon adding the enzyme substrate. In these cases.. or exogenous compounds such as manitol. (A) Direct detection of DNA by electrooxidation of guanine using an electrode poised at a convenient potential.1. uric and ascorbic acid. which oxidises the hydrogen peroxide produced by the enzymatic reaction shown in Fig. Figure 6. other endogenous substances.Sensors 2008. different strategies have contributed to solving the problem. when using an amperometric glucose biosensor. (B) Electrochemical detection of DNA hybridisation using an electrochemical mediator for the oxidation of guanine species. together with the species indicator of the biochemical event taking place at the biorecognition layer. The signal reduces as the analyte concentration increases. e. Labels include electroactive molecules such as ferrocene derivatives [88] (Figure 6B) or enzymes (Figure 6C) [89-91]. labeled oligonucleotide electroactive species target analyte target DNA labeled with a redox-Ez Substrate Product e- capturing probe e- capturing probe e- G C O A G C B C A variety of arrays have been reported to rise above the limitations and disadvantages occurring when using DNA-based biosensors. once hybridisation took place. (C) Electrochemical detection of DNA hybridisation using an oligonucleotide modified with a redox enzyme (Ez).96-98]. Certainly. and some of them will be detailed in the following item. 8 1375 DNA hybridisation. as those referenced next [75. DNA-based biosensor arrangements. sometimes complications crop up using the simple model described in point 2. thiocyanate etc. are also oxidised at the electrode potential [99-103]. as well as other redox-derivative compounds [92-95]. gluthatione.

a cystein-rich protein that covalently binds heavy metals via its multiple sulfhydril residues [116. as described in Fig. A variety of electrochemical mediators. In all these cases. Still another way to remove non-desirable species previously to the detection step is by capturing the analyte. after pairing the target analyte with the biorecognizing element mounted on the beads.118]. These compounds usually act as enzyme (Ez) co-substrates mediating the charge transport between the biological recognition element and the electrode.2-diaminobenzene) [109].Sensors 2008. either soluble or directly attached on the electrode. which can operate based on either charge or size properties.117]. they prevent from electrochemical oxidation/reduction of interferences and. poly(o-phenylenediamine) [112] and cellulose acetate [113. After washing the electrode. In brief. Electrochemical mediators commonly used are chemicals displaying a higher kinetic constant for the electron transfer process at the electrode than that of the product selected to monitor the enzymatic reaction. which can be repetitively transformed. [105]. Magnetic-bead-based biosensors relay on the same principle based on detection of the analyte subsequent to the separative step [119].111]. there is a substantial improvement of the methodology sensitivity due to the recycling of the electrochemically active species. the electro-transformation of the mediator (med) is favoured. and changing the medium where the electrochemical reaction will take place [59. Examples of interference-excluding membranes based upon size used for glucose amperometric biosensors include the dialysis type ones [100. have determined the presence of heavy metals in biological fluids with a biosensor which uses metallothionein. One example of the charge rejection approach has been proposed by Zhang et al. Thus. Most of them deal with the enzyme-electrically-wiring obstacle. as compared with that of the product of the enzymatic reaction. A number of biosensors have been proposed to detect analytes of clinical interest using this strategy. sometimes.114]. such as overoxidized polypyrrol [107]. previously adsorbed on a hanging mercury drop electrode. polymers. poly(1. They designed a blood glucose biosensor that uses a Nafion™ membrane to exclude interfering anions. Another straightforward method to prevent interferences from reaching the electrode is by using a selectively-permeable membrane. Adam et al. polymers allow for permeation of molecules having similar molecular weigh to the analyte. For example.106]. this approach is only useful when dealing with highly sensitive devices. .62. It is worth pointing out that when such a catalytic cycle occurs. interferences of size larger than the analyte being excluded. The second tactic to avoid interfering reactions consists of working with systems that use electrode potentials at which interferences are not efficiently oxidized/reduced. poly(1.116]. such as the one reported to detect uric acid using uricase [104]. have been used.3-diaminobenzene/resorcinol) [110. 7. they still improve sensitivity. the device proposed to detect BRCA1 breast cancer gene being one relevant example [120]. which can be achieved by using electrochemical mediators [121-125]. The procedure consists of capturing the heavy metal ions by their interaction with the metallothionein protein. 8 1376 The simplest proposal to diminish occurrence of non-desired reactions at the electrode is simply by diluting the sample so that the interferences potentially present in the sample are not readily detected. However.115. therefore rendering a catalytic cycle. polydimethylsiloxane [108]. the coupled system is magnetically removed from the original sample matrix and transferred to an appropriate interference-free medium for further detection.60. Simultaneously. a differential pulse voltammetry is performed in a convenient supporting electrolyte to register the corresponding signal [116.

120]. decrease of working potentials.79. as well as other electrode-attached mediators to determine simple molecule analytes. and allowed . Catalytic cycle taking place when using an enzymatic amperometric bioelectrode and an electrochemical mediator that regenerates the original oxidation state of the enzyme redox site. Once more. 1377 analyte product Ez ox Ez red med Red med Ox eelectrode Recently. which is displayed by this Prussian Blue-analogue. severe interferences from oxidation/reduction of other species present in the sample make necessary additional steps to accurately detect hybridisation [56. iron complexes derived from tetra-2-pyridyl-1. dimethyl ferrocene [127. complexes containing Os-bispyridine or derivatives [131-139]. In one of the proposed separative strategies. have described a very interesting mediator for a glucose biosensor [126]. the DNAcapturing probe is attached to magnetic beads and. once hybridisation is accomplished. Proposals of clinically-useful biosensors using soluble mediators to detect infection-marker antibodies [24.Sensors 2008. such as glutathione [132]. among others. Ospoly(allylamine) derivatives [142] and poly(aniline) derivatives [143]. and cholesterol [129] accurately have been also reported. other ferrocene derivatives [129.e.141]. i. lactate [147]. The analyte determination proceeds by electron-transferbased electrochemical techniques [60. working at a relatively low potential [126].128]. Chaubey et al. using copper surfaces [80]. clever improvements to avoid interference reactions and background currents have been proposed when detecting DNA hybridisation. Therefore. inside a highly π-character network. the particles are magnetically removed from the medium. Moreover. as for example. Likewise.59. acetylcholine [145]. Some approaches follow the same strategies as for enzymatic biosensors. have published an extensive study on mediators for biosensor applications [148]. physical separation of the analyte from interferences. poly(allylamine)ferrocene [140. detection of DNA hybridisation by electrocatalytic oxidation of ribose and primary amine residues of nucleotide bases.149-151]. considering that the potentials at which the nucleotide bases are electro-transformed are quite extreme. methyl viologen or derivatives [133.4-pyrazine have been mixed with hexacyanoferrate species to render a supramolecular structure that contains Fe(II) and Fe(III) species.134]. which include tetracyanoquinodimethane. tetrathiafulvalene. Alves et al. L-alanine and pyruvate [10]. This polymer exhibits an excellent conductivity together with catalytic activity for H2O2 electro-reduction. biological oxygen demand [146]. the polymer enables its coupling to a glucose-oxidase enzyme as a redox charge transfer mediator.25]. the ExacTECH™ biosensor from Abbott-Medisense takes advantage of the strategy. Reasonable good performances were also reported for mediators attached on bioelectrodes surface for glucose determination. That has prompted the development of alternative approaches. In this approach ss-DNA signal was lower than that generated by ds-DNA.130]. 8 Figure 7. In this case. toluidine blue O [135]. using in this case one ferrocene derivative as mediator to monitor glucose blood level [144]. or both alternatives at the same time.

2’bipyridyl)3+. mitochondrion. changes were brought up attempting to improve the stability of the immobilized biorecognition element. in which a dialysis membrane kept the bioreceptor. oxygen Nutrients Undamaged tissue From Clark’s first biosensor design. and hence the lifetime of the biosensor. depending on the complexity of the biorecognition element. it has been reported the development of diagnostic biosensors retaining the bioactive molecules via crosslinking with carbodiimide or glutaraldehyde to . in a solution which preserved its integrity. any biological molecule or biomimic assembly capable of recognizing the analyte can be used as a bioreceptor.g. for example.3. Therefore.Sensors 2008.96]. This strategy prevents from working at extreme potentials and it has proved success to detect. Strategies used in biosensor technology to prevent interference reactions from species accompanying the analyte are detailed in reviews [63. Numerous biosensors in which the recognizing elements are entrapped in passive matrices such as inert polymers. glucose oxidase. the requirements to maintain its integrity will change. It is obvious that.155].74. have been described [154. which is easily electro-oxidised at a relatively low potential [152]. Another option to detect hybridisation is to oxidise G via a charge transfer mediator. a sequence of BRCA1 breast cancer gene [153]. for example the metallic complex Ru(2. heavy metals) Mechanic protection Appropriate osmotic pressure. Table 1 summarizes the main needs to be taken into account according to the biorecognition element used in the bioreceptor layer. etc) Cell Tissue Organ Main requests for functional integrity (the needs usually add up to the bottom) Appropriate withholding pH and saline stability Absence of certain poisoning products (e. In principle. 2. 8 1378 to detect nucleic acids in concentration at the picomolar level [80]. or where the analyte-capturing molecule is cross-linked with other spectator proteins. In this line. Most important requirements to preserve the bioreceptor functionality Biorecognition layer (complexity order increases to the bottom) Aptamer / ion carrier (ionophore) Single molecule Antibody / DNA Enzyme / high molecular weigh protein Membrane receptor Organelle (chloroplast. Preserving the bioreceptor layer Bioreceptors must maintain appropriate biorecognition activity to be used in a biosensing device. Table 1.75. their structural/conformational integrity must be conserved so as to display a reasonable lifespan.

(ii) to allow charge propagation and to “wire” the label (e. for example. polyaniline was used to detect antibodies [166]. biocompatibility.g. such as (i) to give a support to the biologically-active molecule by chemical interactions.136. for example. and (iv) to enhance mechanical resistance. Reviews focusing on polymers used to assemble amperometric biosensors are referenced in [172-174]. we will therefore converge on the topic of signal boosting. and creatinine [157]. takes advantage of this polymer and a reasonable stability has been reported [169]. Therefore. Among these polymers. A number of other biosensors for clinical applications using modified surfaces with chitosan polymer have been proposed. In all cases. The main strategies used to diminish the background currents produced by interferences have already been addressed in point 2. For instance. and to determine choline. and in those where glucose oxidase is immobilised in a polypyrrole hybrid film. covalent or non-specific interactions. a biosensor to determine the tumour marker. For example.2. and sensitivity will depend on the natural sample where the device is to be used. dengue virus oligonucleotide sequences [170]. in this latter case the enzyme cholinesterase was cross-linked with glutaraldehyde in a chitosan gel [171]. an enhancement of the signal-to-noise ratio is achieved by increasing the number of electroactive detectable analyte-related species and/or by lowering the interferences that reach the electrode thus giving rise to background current noise. Enhancing any analytical method sensitivity means increasing the signal-to-noise ratio. . whose biorecognition element is an enzyme-labelled antibody. the aimed limit of detection. 8 1379 detect. prostate specific antigen [14]. 3. turning devices more robust. since they render appropriate stability and good conductivity [162].Sensors 2008. transferrin [156]. dynamic range. Two obvious lines of attack are possible: augmenting the signal or diminishing the noise. the expected range of analyte concentration and the interfering substances potentially present in the sample will largely influence the requirements. (iii) to admit the incorporation of redox mediators. either electrostatic. conducting polymers have demonstrated several advantages. Various different bioreceptor-capturing. and appropriate film-forming properties [168]. Other conducting polymers have been used. α-1-fetoprotein. In the last decades.167]. entrapping polymers used to develop clinically suitable biosensors have been described. which has been faced in the manners described below.165]. Schistosoma japonicum antibody [27]. specific chagasic antibodies [25]. to detect Schistosoma japonicum antigen [26]. have been proposed to immobilize biologically-active species because of their advantageous features such as inertness. displaying very good performances [126. Sensitivity One of the most relevant tasks when designing a ready-to-use biosensor is to achieve the needed sensitivity. among others. the pyrrol-based ones have been thoroughly characterised [158-161]. and poly(allylamine) derivatives incorporating a charge transfer mediator have also proved to be efficient for enzyme-based amperometric biosensors [59. one redox-enzyme) to the electrode. Polypyrrole-based polymers have been successfully used in biosensors to detect urea by means of ureasa entrapped in it [163]. natural polymers. In electron transfer-based biosensor technology.164. Among them. the “lowering the noise” subject will not be further detailed. such as chitosan derivatives. They display excellent properties to be used for biosensor development. Hence. good mechanical resistance.

IgG and cocaine [176]. in these cases. Schematic representation of a bi-enzymatic amperometric biosensor. 8).1. one of which is the Ez-2 substrate (Fig. is the substrate of the first enzyme (Ez-1). among others. namely: (i) Recycling the electrochemically-transformed compound. 7). immobilised onto aminopropyl controlled-pore glass [177]. This cyclical . In this approach. two of which are based on magnifying the electron-transfer process. analyte and Ez-1 substrate electroactive species and Ez-1 co-substrate side product Ez-1 product and Ez-2 substrate + Ez-2 Ez-1 + eelectrode Several examples taking advantage of such bi-enzymatic amplification have been described to detect chatecolamines [175. the analyte (or one analyte-related species). Magnifying the electron-transfer process 1380 Different ways of achieving the goal of increasing the number of detectable species are possible. This has already been described above in item 2. The basis of these clinically-relevant biosensors is that one of the products of the first enzymatic reaction is the substrate of the second enzyme. or a polyaniline film [178]. as described previously in point 2. The analyte.176]. which transforms this product back to the initial species. this latter one being electrochemically detected.Sensors 2008. This approach was also used to detect successfully lactate via coupling the enzymes lactate oxidase and lactate dehydrogenase. 8 3. ( ) is recycled as long the electroactive species ( ) is consumed. Therefore. (ii) Amplifying the electron exchange process at the electrode surface using coupled enzymatic systems. The signal that evidences the occurrence of the enzymatic cycle is generally related to the presence (or absence) of the analyte in the sample when performing an indirect (or competitive) immunoassay with electrochemical detection. Ez-2 reacts with its substrate to regenerate the analyte (or the analyte-related species). the enzyme substrate should be present at excess concentration to keep the enzymatic cycle. The analyte and the Ez-1 co-substrate. even tiny amounts of the analyte can be detected because the consumption of the electroactive species is supported by the cyclic reaction of both enzymes.1.2. Usually. which is the electro-active species. Figure 8.2 when considering catalytic enzymatic cycles involving the regeneration of the active-site of the redox enzyme using a charge transfer mediator that is then electrotransformed at the electrode surface (see Fig. react with Ez1 to render the products. thus enabling its recycling to keep consuming Ez-1 co-substrate.

183]. catalyses the reaction between the analyte and maltose. and of parathyroid function [180]. but also by recycling the analyte [179]. to detect inorganic phosphate [179].2. the analyte being the complementary antigen [181. In both . to render β-D-glucose-1-phosphate and α-D-glucose (4). so as to make the analytecapturing step more efficient. the signal is not only amplified by the extra amount of substrate rendered by the acid phosphatase using a “side product”. Increasing the number of biorecognition species The obvious tactic to raise the amount of the electrochemically-detectable species is enlarging the number of biologically-active molecules in the biorecognition layer. which will be also further oxidised according to (6). which can be recycled again. these structures do not offer the desired signal-to-noise ratio and. (iii) Producing additional equivalents of electroactive species. 8 1381 conversion allows one electrochemically-active species. the first enzyme. have demonstrated that the signal can be increased using devices with stratified-analogue structure where polymer layers are alternated with layers of the antibody. In Conrath’s proposal. maltose phosphorylase. Caruso et al. thus releasing the analyte. An amperometric biosensor using this model was presented by Conrath et al. which is the electrochemically detected species. Another enzyme. many times. to keep on being consumed. thus multiplying the measured signal. maltose phosphorylase maltose + Pi mutarotase α-D -glucose + β-D-glucose-1-P (4) α-D-glucose glucose oxidase β-D-glucose (5) β-D-glucose acid phosphatese glucuronic acid + H2O2 electroactive species β-D-glucose + Pi (6) β-D-glucose-1-P (7) In this interesting example. In a similar way. which had rendered the first equivalent of electroactive species. generated by a second enzyme that uses a “side product” of the first enzymatic reaction. phosphate. it has been shown signal enhancement when building up a layer-by-layer. β-D-glucose acts as substrate of glucose oxidase to render glucuronic acid and hydrogen peroxide (6). comprised of a redox polymer and an enzyme-labelled antibody [137.Sensors 2008. hydrolyses the “side product”. 3. In the clinical practice. phosphate levels in both blood and urine are determined as indicative of osteopathies.167. acid phosphatase. therefore. acting as co-substrate of the first enzyme. Self-assembled monolayers have proved to be useful in biosensor design but. organized supramolecular structure. generating an extra β-D-glucose (7). it has been proposed to use multilayer systems.182]. This latter species is further transformed by mutarotase into to β-D-glucose (5). glucose-1-phosphate.

Early studies dealing with this issue were performed by Caruso et al. Indeed. Moreover. Nanomatter is composed of unique functional materials that display incomparable characteristics related with their shape. increased the further-immobilized IgG molecule.e. They demonstrated that electrodes whose surface had been modified with protein A. the calixarene structure has thiol residues which link to the gold surface and.1. Directly related with their arrangement. a molecule that displays high affinity for the Fc region of IgG. and their high surface area per volume unit [188]. nanorods and nanoparticles. i. nanotubes. is named Fc region. the last decade has witnessed the blooming of biosensor technology involving nanomatter [188-191]. oriented attachment onto the activated surface [31]. When self-assembling IgG-based immunoelectrodes. This does not occur in a 2-D . Additionally. Calixarene derivatives have been proposed as linking structures to conduct effective protein immobilization [184]. Thus. our group has reported preliminary results on one biosensor to detect specific chagasic antibodies whose capturing antigen is a genetically modified protein tailored to react oriented with a carbodiimide-activated surface. Using the oriented-linking principle to attach the bioreactive molecules. one-dimension (1-D) structure. but without the selective. a crownether structure remains exposed to further link the analyte-capturing biomolecule [185]. at the opposite side.2. among the most outstanding features of nanomaterials are their mechanical strength. According to their shape. In one proposal. nanosprings. [186]. the so-called Fab region. Sometimes.Sensors 2008. IgG is a Y-shaped molecule. In line with this. enhanced protein binding was achieved by modifying gold electrodes with calixarene-derived monolayers. IgG molecules attach randomly to the surface. it has been possible to build an electron-transfer based biosensor to detect Staphilococcal enterotoxin B [187]. The device has shown sensitivity enhancement as compared with biosensors assembled with the same antigen recognition sites. and only a fraction of them are useful to capture the analyte. electrontransfer process may be largely influenced by charge diminution or accumulation when taking place in a linear. 8 1382 cases. nanomaterials have been classified into nanowires. its base. in a nanowire. Proper oriented attachment is crucial when the bioreactive species comprises a specific IgG antibody. it is desired that the IgG Fab region is favourably oriented so as to prevent blockage of the binding sites to effectively capture the antigen. the remaining extremity of the molecule. whose specific antigen-binding sites are located at the end of the “arms”. the basic principle is that the electrochemical response obtained is proportional to the amount of the analyte-related species captured. correctly oriented to react with its target analyte [186]. In these cases. structure and size (in the order of 1 to 100 nm scale) [192]. In connection with biosensors. or to conduct their attachment in a favourable orientation. The problem is once more tackled by boosting the number of available active sites to interact with the analyte. we could enhance not only the sensitivity but also the selectivity of the method as mentioned above in item 2. a more precise control over surface density of biomolecules to prevent molecular aggregation. their efficient and tuneable electron-transport properties. nanobelts. leads to improved biosensor performances. The work demonstrates that the efficiency to capture the analyte was enhanced when using streptavidin-biotine interactions to favourably orient the bioreceptor attachment [48]. as for example. nanotechnology has emerged as the “star” to achieve the signal-augmentation task. Recently. recent studies on biosensors to determine thrombin have demonstrated the critical importance on the immobilization step.

several strategies have been proposed to modify the nanomaterial surface. this is translated into a plethora of analyte-interacting sites. and that prospects of biosensors useful for clinical diagnosis will be closely related to it. this provides an improved way towards glucose-oxidase biosensors development [200]. On the other hand. When building electrochemical biosensors. charge transport in nanowires or nanotubes takes place along the line direction. by performing separation-free immunoassays [203]. in which metallic electrochemical mediators are used to catalyse G oxidation. have reported that it is feasible to suspend CNT in Nafion solutions. The layout allows spatial separation between the excess of conjugate in solution and the conjugate bound to the bioreceptor . The enzymes glucoseoxidase and horseradish-peroxidase. When charge transport occurs in a 2-D structure. nanomaterials offer massive number of sites that can be chemically functionalized. even those that circumvent high or low charge density sites. which is the only path available to transfer electrons.198]. has been also used together with functionalized carbon nanotubes (CNT) to detect the hybridisation event.193. the use of classical amperometric-based biosensors requires a number of consecutive steps. These amperometric biosensors relay on a self-assembled antibody monolayer attached onto a double-sided microporus gold electrode. the resulting system showing a dramatic decrease of the overpotential for hydrogen peroxide detection. Also gold nanotubes have been modified by alkylthiols to which the enzymes of interest had been attached. In this case. This is an important feature to bear in mind. Thus. Thorough reviews on this subject are referenced as [188-190. such as a virtually planar film. so that the device operation results amenable. generally proceeding according to the methods already established to develop amperometric biosensors. Generally. The vast number of recently published work involving nanotechnology to design electrochemically-based biosensors demonstrates that this is one of the most fertile fields of research. In another example. among others. the already known discriminative properties of Nafion were combined with the recently-demonstrated hydrogen peroxide catalytic properties of CNT [200].193]. gold nanoparticles were utilized to develop amperometric biosensors to determine IgG [194]. have been covalently linked via activation using carbodiimide. For example. 4.202]. for example. The same principle described above when addressing DNA-biosensor technology. 8 1383 structure. the detection limit has been lowered to a few attomoles of the analyte [199]. Simplicity of use and packaging Protocols to be followed when using biosensors should be as simple as possible. Wang et al. commonly used in clinical biosensor technology. It was also demonstrated the feasibility of designing biosensors useful for clinical chemistry analysis by means of CNT-based pastes biocomposite bioelectrodes [201]. thus allowing to determine clinically-relevant analytes [197. and in relation to their high surface-to-volume ratio. Depending on the nanostructure composition. electrons can be transferred along different conducting roads. or cross-linking it with glutaraldehide [197. and lactate [196].198]. Together with this exceptional susceptibility. Clever solutions to shorten procedures have been reported. since it is usually related to market acceptability. thus leading to sensitivity enhancement [188-190].Sensors 2008. hepatitis B surface antigen [195]. This makes these materials competent to give a response upon perturbations from even a few molecules interacting along the line [188.

without using any charge transfer mediator [207].Sensors 2008. Finally. derivatization. the target ss- . since the latter ones reduce production and operative costs. Label-free biosensors have also become attractive because of their easiness of use. The electrode surface was thiolated. Therefore. the proposal has advantages with respect to common ELISA. the apoenzyme was added. the enzyme was effectively reconstituted and glucose transformation into glucuronic acid was then monitored by amperometry. The enzyme co-factor was also covalently attached at the opposite end of the nanotubes by using carbodiimide. sometimes the complexity of the sample carrying the analyte and/or its low concentration make us to face with challenging problems. 5. extraction. thus making the operation not only cheaper than when using macro/semimicro scale techniques but also less hazardous [215]. another attractive characteristic aimed to lower the cost of the analysis [212]. hCG was detected using an immunosensor where Abs against this pregnancy-indicator hormone had been oriented attached by means of protein A. analyte detection [220]. Future directions Even though it is desirable to perform the clinical-chemistry analysis in only one step. The sample diffuses through the pores. Certainly. hCG concentration was determined measuring the peak current by square wave stripping voltammetry [209]. The device consists of an ultramicroelectrode array where a glass chip is integrated with poly(dimethylsiloxane) channels. A good example is the enzymatic biosensor to detect lactate in saliva in a one-step analysis [216]. pre-concentration. The progress in microfluidic systems allows for integration of different steps to perform the analysis within a single package [217-219]. A novel approach has been reported to detect nucleic acids. An electroactive compound is carried inside liposomes labeled with one ss-DNA tag. An outstanding approach has been proposed to determine glucose using glucose-oxidase directly “plugged” to the electrode taking advantage of the controlled electron transference through a CNT [207]. At the present time. The system consists of a silicon wafer where a three-electrode cell is built up. Additional applications where label-free biosensors were used can be found in the following references [210-214].221]. Lactate oxidase is entrapped in an agarose gel placed inside a cavity of the silicon chip. the possibility of working in the micro-scale allows to use few microliters of reagents. The steps may include pretreatments. such as fewer steps and the possibility of performing it in the field [204-206]. In addition. 8 1384 layer. apart from attenuating environmental impact. schemes of biosensors coupled to layouts allowing for separative or pre-concentration steps are promising tools to deal with complex-matrix samples [217]. and the product of the enzymatic reaction is detected by amperometry [216]. Some of them also present renewable devices. eventually. the need to move classical analytical tools into miniaturised devices is easily understood. Micro-scale biosensor devices intrinsically have the ability to react with the analyte and generate a measurable response within the same compact body. In another example. The development of clinically-useful biosensors is moving in that direction due to the fact that the reactant requirements are definitely lower than those of classical methodologies. such as filtration. reaction and. and CNTs were covalently aligned using carbodiimide. The so-called lab-on-a-chip technology has emerged as a gifted tool to cope with these real complex samples [217. Another additional ss-DNA probe is linked to superparamagnetic beads. The reader is referred to a recent review on contacting redox proteins using nanotechnology [208]. which illustrates the matter [222].

Furthermore. i. Marcelo Gabriel Roma . the actual trend in clinical practice to monitor the health-state of the patient is directed towards point-ofcare screening analysis. nanomaterials and microfluidics forefront technology. in such a way that using a single DNA-genosensor may not allow to diagnose the illness. many other alterations occur during the disease. where the electroactive compound is released to be further electrochemically detected [222]. lead to an enormous number of different biomarkers that can be associated with the more than 200 types of cancer [223. temperature control. This illness may be shuttled by a variety of genetic or/and epigentic changes directly acting on the overgrowth capacity of the cells. etc.Sensors 2008.S. most of clinical analysis is carried out in centralised laboratories were high-technology equipment is available. most underserved population in developing countries do not access to state-of-the-art diagnostic methods [229]. the above-mentioned appliances are foreseen as the next-generation diagnostic devices [23]. This is a critical point for treating the disease at an early stage. Acknowledgements CONICET (PIP N° 5303) and ANPCyT (PICTR2002-00057) are acknowledged. and trained personnel perform the determinations under almost ideal conditions.224]. and household equipment [226-228]. which is the case of cancer. accurate and portable systems seems to be an urgent need.224]. Nevertheless. Biosensors for clinical-chemistry analysis have demonstrated to move towards simplifying the analytical procedures with the advantage of multiplexing capability [23]. specific tumor marker that could be used as indicator of every existing type of cancer. Up to these days. multiple variations evidenced as over/under expression of certain proteins or mRNA. This appliance is a good example of compact ultra-micro systems that combine microfluidics and amperometric based biosensors. Nowadays. is another example in reference to biosensor technology as a powerful option to get clinically relevant information. there is not a unique. Considering that samples for clinical analysis are largely complex. these devices can help even when the problem magnifies because not only the sample but also the disease is complex. In such a complex scenario. new tools are necessary to perform simultaneous measurements of various analytes that generate multiple signals at the same time. Up to now. In connection with this. Indeed. using the same strategy already mentioned to determine heavy metal concentration in human fluids [116]. a fact that leads to improved prognosis [23. M. it has been proposed a biosensor to detect one of the most used chemotherapy medication. 8 1385 DNA being complimentary to both tags. For example. A magnet captures the liposome-analyte-magnetic bead sandwich inside the microfluidic channel. work [225]. is expected to greatly help to achieve this goal. Petrlova’s et al. use of standard solutions and calibrators. Additionally. It is worth mentioning that this possibility can provide the physician important information that will facilitate early cancer detection.Belluzo acknowledges a fellowship from CONICET. apart from DNA changes related to cancer. Research on biosensors. interacting synergistically. making possible to reach even rural people who hardly move to Health Centres [24]. biotechnology. cisplatin [225].e. Moreover. it is also important to monitor the anti-tumoral drug concentration in the patient’s fluids as a means to adjust the doses necessary to keep effectiveness of the treatment. the development of such new technology seems reachable. The authors gratefully thank Dr. The emergence of technology that changes sophisticated diagnostic tools by low-cost. Numerous alterations on oncogenes or tumor-suppresor genes may be associated with cancer development.

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