Anal Bioanal Chem (2014) 406:27–33 DOI 10.1007/s00216-013-7459-z
TRENDS
Electrochemical genosensors for the detection of cancer-related miRNAs Susana Campuzano & María Pedrero & José M. Pingarrón
Received: 4 September 2013 / Revised: 22 October 2013 / Accepted: 22 October 2013 / Published online: 19 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract MicroRNAs (miRNAs), new stars of human genetics, are naturally occurring, 19−25 base pair, noncoding RNAs that regulate gene expression posttranscriptionally and have been demonstrated to be excellent biomarkers for cancer diagnosis/prognosis. Because of their short length, sequence similarity, and very low concentration, their detection in real samples is challenging. Among other methods for miRNA detection, electrochemical nucleic acid biosensors exhibit relevant advantages in terms of high sensitivity, ease of use, short assay time, nontoxic experimental steps, and adaptability to point-of-care testing. This article gives a brief overview of recent advances in the rapidly developing area of electrochemical biosensors for miRNA detection. The fundamentals of the different strategies developed to achieve novel signal amplification and sensitive electrochemical detection are discussed, and some examples of relevant approaches are highlighted, along with future prospects and challenges. Keywords Electrochemical . Genosensor . MicroRNA . Cancer
Introduction A microRNA (miRNA) is a naturally occurring small RNA molecule (18−25 nucleotides long), generally single stranded [1], characterized by two-nucleotide 3′ overhanging ends and 5′ phosphate groups [2], and evolutionarily conserved. These molecules act as regulators of protein translation in a broad range of animals, plants, and viruses and control gene expression by a nonperfect pairing of six to eight nucleotides with a S. Campuzano (*) : M. Pedrero : J. M. Pingarrón (*) Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain e-mail: [email protected] e-mail: [email protected] M. Pedrero e-mail: [email protected]
specific target within messenger RNA (mRNA) and subsequent formation of an RNA-induced silencing complex, either inducing the degradation or silencing the translation of their targets mRNAs [3, 4]. Currently, over 1,000 miRNAs have been identified in humans, and these can target more than 30 % of the human genome [4, 5] and are abundant in many human cell types [6]. They work by specifically binding to their targets, so singlenucleotide polymorphisms (SNPs) within the sequence of miRNAs or their target mRNAs can influence the risk of disease and can also be used in the diagnosis of these diseases [7]. As many diseases are caused by the misregulated activity of proteins, miRNAs have also been implicated in a number of diseases, including almost all types of human cancer [8, 9], heart disease, and immunological and neurological diseases. They have critical functions in a variety of biological processes, including development, cell growth, cell differentiation, apoptosis, and tumorigenesis [10]. They also interfere with metastasis, apoptosis, and invasiveness of cancer cells [11]. It is still ambiguous whether the change in the miRNA expression level is the cause or a consequence of the occurrence of cancer [12], but altered expression levels of miRNAs (upregulation or downregulation) have been correlated with cancer type, tumor stage, and response, and they are potentially biomarkers superior to mRNAs for cancer diagnosis, cancer prognosis, and predictive information [7, 13]. Furthermore, being much shorter than mRNAs, miRNAs are less vulnerable to degradation by ribonucleases and, unlike proteins, they are not postsynthetically structurally modified. Although various tissues contribute to the circulating miRNA pool, most miRNAs are probably derived from blood cells [4]. It has been shown that they circulate in blood wrapped in circulating microvesicles called “exosomes” or in a complex with an RNA-binding protein, nucleophosmin 1 (Fig. 1) [4, 14]. Therefore, they are extremely stable and resistant to degradation, thus allowing the detection of miRNA expression patterns directly from blood and other human fluids [7, 9, 15]. Recently, much evidence has emerged that tumor-derived miRNAs are present (at extremely low concentrations,
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Fig. 1 Circulating miRNAs contained within vesicles (exosomes, microparticles, apoptotic bodies) in protein complexes [argonaute 2 (Ago2), nucleophosmin 1 (NPM1)] and in lipoprotein complexes (highdensity lipoprotein and low-density lipoprotein complexes). (Reprinted from [44] with permission)
typically less than picomolar concentrations) and detectable in serum, plasma, urine, and other human body fluids. Because of their abundance, tissue specificity, and relative stability, circulating miRNAs hold great promise as noninvasive or minimally invasive biomarkers for cancer [15]. However, their small size, sequence homology among family members, and low abundance in total RNA samples make detection of miRNAs challenging [16]. The small size of miRNAs, similarly to the primers used in conventional polymerase chain reaction (PCR), greatly complicates the assays based on PCR or hybridization. In the first case, very short primers would be required, which affects the PCR efficiency because of a very low melting temperature [4]. For hybridization-based detection, it is difficult to label the short probe for selective detection of miRNAs, and the lengths of the capture and detector probes for the development of sandwich detection methods are also limited [17]. Further, the melting temperature of the duplex formed by the probe and its target is low, which sharply decreases the stringency of hybridization and increases the risk of cross-hybridization [4]. Moreover, the similarity in the sequences also makes hybridization-based detection methods difficult. As a result, methods able to include SNP assays are preferred for identifying and quantifying miRNAs [18]. Furthermore, miRNAs represent only a small fraction (approximately 0.01 %) of the total RNA sample; the miRNA concentration can be as low as
a few molecules per cell [4]. Also, for in situ detection, when a sample is a mixture of precursor miRNA and mature miRNA, the oligonucleotide probe can hybridize nonspecifically to precursor miRNA, which can cause a false-positive signal for expression levels of the functional mature miRNA [17]. From these comments, it is important and urgent to develop innovative analytical tools able to couple high sensitivity and specificity for the rapid detection of miRNAs in cells, tissues, or body fluids, and also to be able to distinguish between the precursor and mature forms of an miRNA [15, 17]. Two other important considerations include dynamic range and multiplexing capability. The expression level of miRNAs ranges by as much as four orders of magnitude, from a few copies to over 50,000 copies per cell; thus, a wide dynamic range of detection is required for miRNA assays. Moreover, a single gene can be simultaneously regulated by multiple miRNAs and multiple miRNAs can be simultaneously misregulated, which require methods to detect multiple miRNAs to fully understand the importance and complex function of these tiny regulators and to define a particular disease state [4, 19]. The standard methods for detection of miRNAs are Northern blotting and in situ hybridization, which have low sensitivity and require many steps, resulting in laborious, time-consuming procedures that are difficult to implement for routine miRNA analysis [15, 17, 20, 21]. Alternative methods include oligonucleotide microarrays, quantitative reverse-transcription PCR,
Electrochemical genosensors for cancer-related miRNAs
deep sequencing, surface-enhanced Raman scattering, surface plasmon resonance, surface plasmon resonance imaging, nanoparticle-based or conducting-polymer-nanowire-based fluorescence, and bioluminescence-based techniques. However, many of these methods are long and laborious, some involve amplification reactions, and others require the use of a wellequipped laboratory with specialized and well-trained personnel, and are neither feasible for routine determination of miRNAs nor applicable for point-of-care (POC) testing [18, 22, 23]. A great deal of effort, therefore, is being devoted to developing rapid, simple, highly sensitive, selective, reliable, and low-cost analytical methods that can effectively profile miRNAs in minimal amounts of sample. Compared with the aforementioned methods, electrochemical DNA hybridization sensors, also known as genosensors, hold great promise to serve as devices suitable for POC diagnostics and multiplexed platforms for sensitive, specific, fast, simple, low-cost, and potentially decentralized analysis of nucleic acids in lowvolume samples (from several microliters to hundreds of nanoliters) [24]. They can achieve sensitivities down to several femtomoles per liter (in some cases attomoles per liter) for short oligonucleotides without PCR sample amplification, which is very attractive for detection of low-abundance miRNA molecules [25, 26], and also a major advantage for POC systems. The stringency of electrochemically controlled DNA recognition makes possible single-base mismatch specificity, even in clinical samples. Genosensors have excellent compatibility with advanced semiconductor technology and can be integrated with sample preparation and fluidic processes, making possible rapid, multiplexed nucleic acid detection for portable POC clinical diagnostics. Advancements in microtechnologies and nanotechnologies, specifically fabrication techniques and new nanomaterials, are largely responsible for the development of highly sensitive and specific electrochemical sensors, making them suitable for the detection of miRNAs (Fig. 2). In particular, the detection sensitivity is enhanced through highly specific molecular recognition (via appropriately designed targets and probes), improved electrochemical signal generation, transduction, and amplification, and enhanced electrical conductivity for minimized background noise [24]. In the following sections, the rapid progress of cancerrelated-miRNA electrochemical genosensors will be addressed, with an overview of recent efforts in this area, and highlighting two examples of elegant sensing concepts. Finally, future developments and research directions in this field are also discussed.
Electrochemical genosensors for miRNAs: state of the art Sensitive electrochemical biosensors for detection of miRNAs based on miRNAs labeled with electrocatalytic moieties [27]
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or nanoparticle tags [5, 6, 8, 21, 28, 29] have been developed. However, these detection methods need to enrich and label miRNAs, which increases the complexity of the operation because of the low abundance of miRNAs in real samples. Therefore, label-free electrochemical biosensors have attracted attention in order to increase the simplicity [8]. They involve the direct [22] or catalyzed [30] oxidation of RNA bases, as well as redox reactions of reporter molecules [31, 32], enzymes [12, 13, 29, 33–35], and viral proteins [1, 12, 14] recruited to the electrode surface by specific RNA probe–target interactions. Other approaches involved nanostructuring of electrode surfaces with networks of carbon nanomaterials and electroactive polymers [36], conducting polymer nanowires [37], and graphene and dendritic gold nanostructures combined with multifunctional encoded DNA–gold nanoparticle (AuNP)–locked nucleic acid (LNA) bio-barcodes [29]. The nanoscale sizes of these nanomaterials break through the limitation of structure miniaturization, and result in low detection limits [4]. The short lengths of miRNAs with inherently different melting temperatures and the highly similar sequences among miRNA family members make probe design more difficult than for mRNA arrays [5]. To minimize the risk of crosshybridization and achieve high detection sensitivity, alternative probes have been used to increase the melting temperature of probe–miRNA heteroduplexes [12]. Up to now, the probes applied in biosensors for miRNA detection have been mainly DNA [13, 22], hairpin [32], peptide nucleic acid [31, 33, 38, 39], and LNA [8, 29, 40] capture probes. An interesting approach worth highlighting is the threemode electrochemical biosensor for miRNA detection developed by Labib et al. [14] that brings together the advantages of electrochemical genosensors and the unique binding property of p19 protein (from Carnation Italian ringspot virus) toward small 21–23-bp double-stranded RNA with nanomolar affinity. The p19 protein behaves like a molecular caliper of double-stranded RNA and sequesters miRNAs in a sizedependent, sequence-independent manner [1]. The sensor, based on the self-assembly of a thiolated RNA onto a AuNP-modified screen-printed carbon electrode, is able to detect one or multiple miRNAs using three detection modalities based on hybridization, p19 protein binding, and protein displacement (see Fig. 3). The hybridization of the target miRNA to its complementary immobilized probe causes an increase in the current, measured by square wave voltammetry (Fig. 3, step b). Furthermore, addition of the p19 protein dimer to the hybrid formed causes a large decrease in current density, thus amplifying the signal (Fig. 3, step c). A universal displacement-based sensor (Fig. 3, step d) is formed on the basis of the self-assembled thiolated RNA probe bound to a saturated concentration of an miRNA, whereas p19 is attached to the hybrid formed. Subsequently, a mixture of a target miRNA and a nonthiolated RNA probe is incubated with the
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Fig. 2 The integration of nanomaterials and microfabrication/nanofabrication technologies for the construction of electrochemical DNA sensors. MEMS microelectromechanical system. (Reprinted from [24] with permission)
p19-modified sensor. The newly formed hybrid in solution, at relatively high concentration compared with that of the immobilized hybrid, can force p19 to dissociate from the immobilized hybrid on the electrode surface and to bind to the newly formed hybrid, causing a shift-back of the signal. Uniquely, the protein displacement mode allows the detection of any type of miRNA without using thiolated probes. The three sensing modalities combined in one sensor link high sensitivity (5 aM miRNA or 90 molecules of miRNA per 30 μL of sample without PCR amplification), a broad dynamic range of measured concentrations (11 orders of magnitude, from 10 aM to 1 μM), triple verification of the miRNA concentration, and sequential analysis of two different miRNAs on one electrode. In addition, this sensor can recognize miRNAs with different A/U and G/C contents and detect SNPs. It has been successfully used and validated by Fig. 3 The three-mode electrochemical sensor for detection of miRNAs. GNPsSPCE gold-nanoparticlemodified screen-printed carbon electrode. (Reprinted from [14] with permission)
quantitative real-time PCR for direct detection and profiling of three endogenous miRNAs in human serum—hsa-miR-21, hsa-miR-32, and hsa-miR-122—which have been reported as biomarkers for colorectal, prostate, and liver tumors, respectively. The single use of the sensors developed opens a new avenue for large-scale production of disposable miRNA diagnostics with significant impact on early detection of diseases using biological fluids. Another remarkable approach is that developed by Yin et al. [29] for direct detection of miRNA hybridization without a label and enrichment based on triple-signal amplification (Fig. 4). Graphene nanosheets and dendritic gold nanostructures were immobilized onto a glassy carbon electrode to improve the electrochemical effective surface area to immobilize a larger amount of an LNA capture probe. In a further step, AuNP-functionalized DNA bio-barcodes were
Electrochemical genosensors for cancer-related miRNAs
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Fig. 4 Chronoamperometric determination of miRNA hybridization using three amplification steps. AuNPs gold nanoparticles, GCE glassy carbon electrode, HRP horseradish peroxidase, LNA locked nucleic acid, MB molecular beacon. (Reprinted from [29] with permission)
hybridized with the 3′ end of the probe to increase the immobilization amount of biotin. Then, streptavidin–horseradish peroxidase specifically interacted with biotin to amplify the electrochemical response of benzoquinone. On the basis of these amplifications, a detection limit of 0.06 pM was achieved. This highly sensitive and selective biosensor allowed the direct and PCR-free analysis of miR-21 expression in total RNA extracted from human hepatocarcinoma BEL-7402 cells. Studies of miRNA expression levels performed using these electrochemical genosensors exhibited good agreement with the common Northern blotting, while being more efficient and more sensitive. Furthermore, the methods developed are simpler and do not require PCR, labeling with toxic substances, sophisticated and expensive devices, large-volume samples, complex sample pretreatment, or analyte enrichment [41]. Some of the biosensors implemented are reusable [14, 18] and repeated assays can be performed at a single electrode for
multiple samples, drastically increasing the sample throughput of the method and reducing the cost and time per assay. In these approaches, multiple electrodes can be prepared concurrently for assays of many different samples, also enhancing the fidelity and repeatability of replicate sample measurements. The characteristics of these novel electrochemical miRNA biosensors make them well suited for the detection of these specific biomarkers in complex body fluids such as saliva and serum and constitute significant advances toward routine detection of miRNAs in both clinical and research settings.
Outlook Use of miRNAs as cancer biomarkers has become a glimmer of hope for the early diagnosis of this fatal disease. As a consequence, the detection of circulating miRNAs in body fluids is emerging as a promising novel tool to improve cancer
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screening and assessment of disease progression, thus making possible tailored treatment. Although the field of circulating miRNA research is still in its infancy, it shows great potential since some miRNAs are abundant, are tissue-specific, are relatively stable molecules, are easily accessible, and can be collected in a relatively noninvasive manner. As we continue to discover the vast impact that miRNAs have on the course of diseases, such as cancer, and the immense promise they hold in the area of gene therapy, rapid and sensitive methods for their direct detection and quantification are of utmost importance. An effective method for miRNA profiling should (1) involve easy and rapid experimental protocols, (2) require the minimum sample quantity, (3) have a high specificity and sensitivity with a large measurement dynamic range from subfemtomolar to nanomolar concentrations, and (4) have low cost. Even if this ideal technique does not yet exist, all the electrochemical miRNA biosensors developed until now address these issues through various strategies: signal amplification through enzyme substrate turnover, highly favorable hybridization conditions using LNA or peptide nucleic acid capture probes, optimized reporter molecules such as AuNPs, or highly tuned and responsive electrocatalytic/amperometric monitoring. Nowadays, methods using advanced electrochemical genosensors stand out above the current “gold standards” because they achieve comparable or improved sensitivity and specificity without the necessity for potentially hazardous materials (radioactive labels or toxic dyes) and highly expensive imaging equipment. These factors are becoming increasingly important in today’s environmentally conscious society, and by drastically reducing the procedural complexity and overall expense of such assays, they make research in the field of miRNAs much more accessible. The unique and attractive strengths of electrochemical genosensors are extremely promising for improving the efficiency of diagnostic testing and therapy monitoring with the construction of multiplexed arrays. Clearly, research efforts in electrochemical detection of miRNAs have achieved the necessary ultrahigh sensitivity and selectivity with the potential for multiplexing and adoptability to POC testing and hence are expected to contribute immensely to the early detection and accurate prognosis of cancers. Typical levels of circulating miRNAs in serum were estimated to be in the 200 aM to 20 pM range [42]. Although many electrochemical biosensors developed for miRNA detection allow detection limits in the low picomolar range to be achieved [1, 10, 43], very recently ultrasensitive miRNA detection (at the attomolar level) has been reported using DNA four-way junction [42] and DNA–Au bio-barcode [29, 43] amplification strategies. With multiple alternatives for ultrahigh sensitive electrochemical genosensors and the beginnings of translation to multiRNA arrays, the first few big steps along the road to electrochemical arrays for detection and monitoring of
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cancer-related miRNAs have been taken. However, there are still several issues which must be addressed before they are accepted among the current standard PCR or Northern blotting methods. Generally, the methods must be more robust before they can be used as standard procedures and must be adaptable to multiplex miRNA detection platforms. Furthermore, there a few reports in which these highly sensitive electrochemical genosensors have been validated using complex biological or real patient samples. These are the kinds of challenges the assays discussed will face as they are developed further. Little attention has been also paid to the long-term storage stability of electrochemical genosensors for miRNA detection, which is of the utmost importance in routine and real diagnostic applications. In this sense, a rational optimization of surface chemistry involving the use of multicomponent self-assembled monolayers would lead to the design of highly stable miRNA interfaces involving common chemicals and obviating the need for specialized expensive reagents. The need for ultrasensitive assay of low-abundance miRNAs and the trend toward miniaturized devices has made nanomaterials significant, since they can produce a synergic effect between catalytic activity, conductivity, and biocompatibility to accelerate the signal transduction, leading to ultrasensitive detection. However, before their application for clinical diagnosis, these promising nanotechnology-based approaches must be fully validated critically for predictive sensitivity and selectivity by using patient samples. The probability of obtaining false-positive/false-negative results and impact of nanomaterials on humans and the environment should be evaluated. In these nanomaterial-based approaches, significant work needs to be devoted to the simplicity and time required for fabrication/modification, biocompatibility, and environmental friendliness. In particular, better understanding of the nanostructuring protocols will aim at guiding and tailoring the so-modified electrode platforms to meet the needs of specific applications. Furthermore, to increase the biocompatibility of nanomaterials, a significant direction to explore is the use of mild biofunctional ways for the attachment of the biomolecules on the surface of the nanostructures. Some other significant challenges involve reliable microarray fabrication, integration into automated systems (essential to achieve widespread POC use), optimization with real biological samples, and increased speed of analysis. POC testing should be fast enough to relay results quickly, leading to an improved prognosis and aiding in surgical decisions. In summary, there are significant challenges to meet before POC electrochemical arrays for cancer diagnosis through detection of miRNA expression profiles become a reality. The effort devoted by the scientific community in this research field is unprecedented, allowing a certain optimism for the years to come, in which the introduction of these miRNAs and the identification of novel ones in clinical practice seems about to become reality.
Electrochemical genosensors for cancer-related miRNAs Acknowledgments The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ1642) is gratefully acknowledged.
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TRENDS
Electrochemical genosensors for the detection of cancer-related miRNAs Susana Campuzano & María Pedrero & José M. Pingarrón
Received: 4 September 2013 / Revised: 22 October 2013 / Accepted: 22 October 2013 / Published online: 19 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract MicroRNAs (miRNAs), new stars of human genetics, are naturally occurring, 19−25 base pair, noncoding RNAs that regulate gene expression posttranscriptionally and have been demonstrated to be excellent biomarkers for cancer diagnosis/prognosis. Because of their short length, sequence similarity, and very low concentration, their detection in real samples is challenging. Among other methods for miRNA detection, electrochemical nucleic acid biosensors exhibit relevant advantages in terms of high sensitivity, ease of use, short assay time, nontoxic experimental steps, and adaptability to point-of-care testing. This article gives a brief overview of recent advances in the rapidly developing area of electrochemical biosensors for miRNA detection. The fundamentals of the different strategies developed to achieve novel signal amplification and sensitive electrochemical detection are discussed, and some examples of relevant approaches are highlighted, along with future prospects and challenges. Keywords Electrochemical . Genosensor . MicroRNA . Cancer
Introduction A microRNA (miRNA) is a naturally occurring small RNA molecule (18−25 nucleotides long), generally single stranded [1], characterized by two-nucleotide 3′ overhanging ends and 5′ phosphate groups [2], and evolutionarily conserved. These molecules act as regulators of protein translation in a broad range of animals, plants, and viruses and control gene expression by a nonperfect pairing of six to eight nucleotides with a S. Campuzano (*) : M. Pedrero : J. M. Pingarrón (*) Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain e-mail: [email protected] e-mail: [email protected] M. Pedrero e-mail: [email protected]
specific target within messenger RNA (mRNA) and subsequent formation of an RNA-induced silencing complex, either inducing the degradation or silencing the translation of their targets mRNAs [3, 4]. Currently, over 1,000 miRNAs have been identified in humans, and these can target more than 30 % of the human genome [4, 5] and are abundant in many human cell types [6]. They work by specifically binding to their targets, so singlenucleotide polymorphisms (SNPs) within the sequence of miRNAs or their target mRNAs can influence the risk of disease and can also be used in the diagnosis of these diseases [7]. As many diseases are caused by the misregulated activity of proteins, miRNAs have also been implicated in a number of diseases, including almost all types of human cancer [8, 9], heart disease, and immunological and neurological diseases. They have critical functions in a variety of biological processes, including development, cell growth, cell differentiation, apoptosis, and tumorigenesis [10]. They also interfere with metastasis, apoptosis, and invasiveness of cancer cells [11]. It is still ambiguous whether the change in the miRNA expression level is the cause or a consequence of the occurrence of cancer [12], but altered expression levels of miRNAs (upregulation or downregulation) have been correlated with cancer type, tumor stage, and response, and they are potentially biomarkers superior to mRNAs for cancer diagnosis, cancer prognosis, and predictive information [7, 13]. Furthermore, being much shorter than mRNAs, miRNAs are less vulnerable to degradation by ribonucleases and, unlike proteins, they are not postsynthetically structurally modified. Although various tissues contribute to the circulating miRNA pool, most miRNAs are probably derived from blood cells [4]. It has been shown that they circulate in blood wrapped in circulating microvesicles called “exosomes” or in a complex with an RNA-binding protein, nucleophosmin 1 (Fig. 1) [4, 14]. Therefore, they are extremely stable and resistant to degradation, thus allowing the detection of miRNA expression patterns directly from blood and other human fluids [7, 9, 15]. Recently, much evidence has emerged that tumor-derived miRNAs are present (at extremely low concentrations,
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Fig. 1 Circulating miRNAs contained within vesicles (exosomes, microparticles, apoptotic bodies) in protein complexes [argonaute 2 (Ago2), nucleophosmin 1 (NPM1)] and in lipoprotein complexes (highdensity lipoprotein and low-density lipoprotein complexes). (Reprinted from [44] with permission)
typically less than picomolar concentrations) and detectable in serum, plasma, urine, and other human body fluids. Because of their abundance, tissue specificity, and relative stability, circulating miRNAs hold great promise as noninvasive or minimally invasive biomarkers for cancer [15]. However, their small size, sequence homology among family members, and low abundance in total RNA samples make detection of miRNAs challenging [16]. The small size of miRNAs, similarly to the primers used in conventional polymerase chain reaction (PCR), greatly complicates the assays based on PCR or hybridization. In the first case, very short primers would be required, which affects the PCR efficiency because of a very low melting temperature [4]. For hybridization-based detection, it is difficult to label the short probe for selective detection of miRNAs, and the lengths of the capture and detector probes for the development of sandwich detection methods are also limited [17]. Further, the melting temperature of the duplex formed by the probe and its target is low, which sharply decreases the stringency of hybridization and increases the risk of cross-hybridization [4]. Moreover, the similarity in the sequences also makes hybridization-based detection methods difficult. As a result, methods able to include SNP assays are preferred for identifying and quantifying miRNAs [18]. Furthermore, miRNAs represent only a small fraction (approximately 0.01 %) of the total RNA sample; the miRNA concentration can be as low as
a few molecules per cell [4]. Also, for in situ detection, when a sample is a mixture of precursor miRNA and mature miRNA, the oligonucleotide probe can hybridize nonspecifically to precursor miRNA, which can cause a false-positive signal for expression levels of the functional mature miRNA [17]. From these comments, it is important and urgent to develop innovative analytical tools able to couple high sensitivity and specificity for the rapid detection of miRNAs in cells, tissues, or body fluids, and also to be able to distinguish between the precursor and mature forms of an miRNA [15, 17]. Two other important considerations include dynamic range and multiplexing capability. The expression level of miRNAs ranges by as much as four orders of magnitude, from a few copies to over 50,000 copies per cell; thus, a wide dynamic range of detection is required for miRNA assays. Moreover, a single gene can be simultaneously regulated by multiple miRNAs and multiple miRNAs can be simultaneously misregulated, which require methods to detect multiple miRNAs to fully understand the importance and complex function of these tiny regulators and to define a particular disease state [4, 19]. The standard methods for detection of miRNAs are Northern blotting and in situ hybridization, which have low sensitivity and require many steps, resulting in laborious, time-consuming procedures that are difficult to implement for routine miRNA analysis [15, 17, 20, 21]. Alternative methods include oligonucleotide microarrays, quantitative reverse-transcription PCR,
Electrochemical genosensors for cancer-related miRNAs
deep sequencing, surface-enhanced Raman scattering, surface plasmon resonance, surface plasmon resonance imaging, nanoparticle-based or conducting-polymer-nanowire-based fluorescence, and bioluminescence-based techniques. However, many of these methods are long and laborious, some involve amplification reactions, and others require the use of a wellequipped laboratory with specialized and well-trained personnel, and are neither feasible for routine determination of miRNAs nor applicable for point-of-care (POC) testing [18, 22, 23]. A great deal of effort, therefore, is being devoted to developing rapid, simple, highly sensitive, selective, reliable, and low-cost analytical methods that can effectively profile miRNAs in minimal amounts of sample. Compared with the aforementioned methods, electrochemical DNA hybridization sensors, also known as genosensors, hold great promise to serve as devices suitable for POC diagnostics and multiplexed platforms for sensitive, specific, fast, simple, low-cost, and potentially decentralized analysis of nucleic acids in lowvolume samples (from several microliters to hundreds of nanoliters) [24]. They can achieve sensitivities down to several femtomoles per liter (in some cases attomoles per liter) for short oligonucleotides without PCR sample amplification, which is very attractive for detection of low-abundance miRNA molecules [25, 26], and also a major advantage for POC systems. The stringency of electrochemically controlled DNA recognition makes possible single-base mismatch specificity, even in clinical samples. Genosensors have excellent compatibility with advanced semiconductor technology and can be integrated with sample preparation and fluidic processes, making possible rapid, multiplexed nucleic acid detection for portable POC clinical diagnostics. Advancements in microtechnologies and nanotechnologies, specifically fabrication techniques and new nanomaterials, are largely responsible for the development of highly sensitive and specific electrochemical sensors, making them suitable for the detection of miRNAs (Fig. 2). In particular, the detection sensitivity is enhanced through highly specific molecular recognition (via appropriately designed targets and probes), improved electrochemical signal generation, transduction, and amplification, and enhanced electrical conductivity for minimized background noise [24]. In the following sections, the rapid progress of cancerrelated-miRNA electrochemical genosensors will be addressed, with an overview of recent efforts in this area, and highlighting two examples of elegant sensing concepts. Finally, future developments and research directions in this field are also discussed.
Electrochemical genosensors for miRNAs: state of the art Sensitive electrochemical biosensors for detection of miRNAs based on miRNAs labeled with electrocatalytic moieties [27]
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or nanoparticle tags [5, 6, 8, 21, 28, 29] have been developed. However, these detection methods need to enrich and label miRNAs, which increases the complexity of the operation because of the low abundance of miRNAs in real samples. Therefore, label-free electrochemical biosensors have attracted attention in order to increase the simplicity [8]. They involve the direct [22] or catalyzed [30] oxidation of RNA bases, as well as redox reactions of reporter molecules [31, 32], enzymes [12, 13, 29, 33–35], and viral proteins [1, 12, 14] recruited to the electrode surface by specific RNA probe–target interactions. Other approaches involved nanostructuring of electrode surfaces with networks of carbon nanomaterials and electroactive polymers [36], conducting polymer nanowires [37], and graphene and dendritic gold nanostructures combined with multifunctional encoded DNA–gold nanoparticle (AuNP)–locked nucleic acid (LNA) bio-barcodes [29]. The nanoscale sizes of these nanomaterials break through the limitation of structure miniaturization, and result in low detection limits [4]. The short lengths of miRNAs with inherently different melting temperatures and the highly similar sequences among miRNA family members make probe design more difficult than for mRNA arrays [5]. To minimize the risk of crosshybridization and achieve high detection sensitivity, alternative probes have been used to increase the melting temperature of probe–miRNA heteroduplexes [12]. Up to now, the probes applied in biosensors for miRNA detection have been mainly DNA [13, 22], hairpin [32], peptide nucleic acid [31, 33, 38, 39], and LNA [8, 29, 40] capture probes. An interesting approach worth highlighting is the threemode electrochemical biosensor for miRNA detection developed by Labib et al. [14] that brings together the advantages of electrochemical genosensors and the unique binding property of p19 protein (from Carnation Italian ringspot virus) toward small 21–23-bp double-stranded RNA with nanomolar affinity. The p19 protein behaves like a molecular caliper of double-stranded RNA and sequesters miRNAs in a sizedependent, sequence-independent manner [1]. The sensor, based on the self-assembly of a thiolated RNA onto a AuNP-modified screen-printed carbon electrode, is able to detect one or multiple miRNAs using three detection modalities based on hybridization, p19 protein binding, and protein displacement (see Fig. 3). The hybridization of the target miRNA to its complementary immobilized probe causes an increase in the current, measured by square wave voltammetry (Fig. 3, step b). Furthermore, addition of the p19 protein dimer to the hybrid formed causes a large decrease in current density, thus amplifying the signal (Fig. 3, step c). A universal displacement-based sensor (Fig. 3, step d) is formed on the basis of the self-assembled thiolated RNA probe bound to a saturated concentration of an miRNA, whereas p19 is attached to the hybrid formed. Subsequently, a mixture of a target miRNA and a nonthiolated RNA probe is incubated with the
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Fig. 2 The integration of nanomaterials and microfabrication/nanofabrication technologies for the construction of electrochemical DNA sensors. MEMS microelectromechanical system. (Reprinted from [24] with permission)
p19-modified sensor. The newly formed hybrid in solution, at relatively high concentration compared with that of the immobilized hybrid, can force p19 to dissociate from the immobilized hybrid on the electrode surface and to bind to the newly formed hybrid, causing a shift-back of the signal. Uniquely, the protein displacement mode allows the detection of any type of miRNA without using thiolated probes. The three sensing modalities combined in one sensor link high sensitivity (5 aM miRNA or 90 molecules of miRNA per 30 μL of sample without PCR amplification), a broad dynamic range of measured concentrations (11 orders of magnitude, from 10 aM to 1 μM), triple verification of the miRNA concentration, and sequential analysis of two different miRNAs on one electrode. In addition, this sensor can recognize miRNAs with different A/U and G/C contents and detect SNPs. It has been successfully used and validated by Fig. 3 The three-mode electrochemical sensor for detection of miRNAs. GNPsSPCE gold-nanoparticlemodified screen-printed carbon electrode. (Reprinted from [14] with permission)
quantitative real-time PCR for direct detection and profiling of three endogenous miRNAs in human serum—hsa-miR-21, hsa-miR-32, and hsa-miR-122—which have been reported as biomarkers for colorectal, prostate, and liver tumors, respectively. The single use of the sensors developed opens a new avenue for large-scale production of disposable miRNA diagnostics with significant impact on early detection of diseases using biological fluids. Another remarkable approach is that developed by Yin et al. [29] for direct detection of miRNA hybridization without a label and enrichment based on triple-signal amplification (Fig. 4). Graphene nanosheets and dendritic gold nanostructures were immobilized onto a glassy carbon electrode to improve the electrochemical effective surface area to immobilize a larger amount of an LNA capture probe. In a further step, AuNP-functionalized DNA bio-barcodes were
Electrochemical genosensors for cancer-related miRNAs
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Fig. 4 Chronoamperometric determination of miRNA hybridization using three amplification steps. AuNPs gold nanoparticles, GCE glassy carbon electrode, HRP horseradish peroxidase, LNA locked nucleic acid, MB molecular beacon. (Reprinted from [29] with permission)
hybridized with the 3′ end of the probe to increase the immobilization amount of biotin. Then, streptavidin–horseradish peroxidase specifically interacted with biotin to amplify the electrochemical response of benzoquinone. On the basis of these amplifications, a detection limit of 0.06 pM was achieved. This highly sensitive and selective biosensor allowed the direct and PCR-free analysis of miR-21 expression in total RNA extracted from human hepatocarcinoma BEL-7402 cells. Studies of miRNA expression levels performed using these electrochemical genosensors exhibited good agreement with the common Northern blotting, while being more efficient and more sensitive. Furthermore, the methods developed are simpler and do not require PCR, labeling with toxic substances, sophisticated and expensive devices, large-volume samples, complex sample pretreatment, or analyte enrichment [41]. Some of the biosensors implemented are reusable [14, 18] and repeated assays can be performed at a single electrode for
multiple samples, drastically increasing the sample throughput of the method and reducing the cost and time per assay. In these approaches, multiple electrodes can be prepared concurrently for assays of many different samples, also enhancing the fidelity and repeatability of replicate sample measurements. The characteristics of these novel electrochemical miRNA biosensors make them well suited for the detection of these specific biomarkers in complex body fluids such as saliva and serum and constitute significant advances toward routine detection of miRNAs in both clinical and research settings.
Outlook Use of miRNAs as cancer biomarkers has become a glimmer of hope for the early diagnosis of this fatal disease. As a consequence, the detection of circulating miRNAs in body fluids is emerging as a promising novel tool to improve cancer
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screening and assessment of disease progression, thus making possible tailored treatment. Although the field of circulating miRNA research is still in its infancy, it shows great potential since some miRNAs are abundant, are tissue-specific, are relatively stable molecules, are easily accessible, and can be collected in a relatively noninvasive manner. As we continue to discover the vast impact that miRNAs have on the course of diseases, such as cancer, and the immense promise they hold in the area of gene therapy, rapid and sensitive methods for their direct detection and quantification are of utmost importance. An effective method for miRNA profiling should (1) involve easy and rapid experimental protocols, (2) require the minimum sample quantity, (3) have a high specificity and sensitivity with a large measurement dynamic range from subfemtomolar to nanomolar concentrations, and (4) have low cost. Even if this ideal technique does not yet exist, all the electrochemical miRNA biosensors developed until now address these issues through various strategies: signal amplification through enzyme substrate turnover, highly favorable hybridization conditions using LNA or peptide nucleic acid capture probes, optimized reporter molecules such as AuNPs, or highly tuned and responsive electrocatalytic/amperometric monitoring. Nowadays, methods using advanced electrochemical genosensors stand out above the current “gold standards” because they achieve comparable or improved sensitivity and specificity without the necessity for potentially hazardous materials (radioactive labels or toxic dyes) and highly expensive imaging equipment. These factors are becoming increasingly important in today’s environmentally conscious society, and by drastically reducing the procedural complexity and overall expense of such assays, they make research in the field of miRNAs much more accessible. The unique and attractive strengths of electrochemical genosensors are extremely promising for improving the efficiency of diagnostic testing and therapy monitoring with the construction of multiplexed arrays. Clearly, research efforts in electrochemical detection of miRNAs have achieved the necessary ultrahigh sensitivity and selectivity with the potential for multiplexing and adoptability to POC testing and hence are expected to contribute immensely to the early detection and accurate prognosis of cancers. Typical levels of circulating miRNAs in serum were estimated to be in the 200 aM to 20 pM range [42]. Although many electrochemical biosensors developed for miRNA detection allow detection limits in the low picomolar range to be achieved [1, 10, 43], very recently ultrasensitive miRNA detection (at the attomolar level) has been reported using DNA four-way junction [42] and DNA–Au bio-barcode [29, 43] amplification strategies. With multiple alternatives for ultrahigh sensitive electrochemical genosensors and the beginnings of translation to multiRNA arrays, the first few big steps along the road to electrochemical arrays for detection and monitoring of
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cancer-related miRNAs have been taken. However, there are still several issues which must be addressed before they are accepted among the current standard PCR or Northern blotting methods. Generally, the methods must be more robust before they can be used as standard procedures and must be adaptable to multiplex miRNA detection platforms. Furthermore, there a few reports in which these highly sensitive electrochemical genosensors have been validated using complex biological or real patient samples. These are the kinds of challenges the assays discussed will face as they are developed further. Little attention has been also paid to the long-term storage stability of electrochemical genosensors for miRNA detection, which is of the utmost importance in routine and real diagnostic applications. In this sense, a rational optimization of surface chemistry involving the use of multicomponent self-assembled monolayers would lead to the design of highly stable miRNA interfaces involving common chemicals and obviating the need for specialized expensive reagents. The need for ultrasensitive assay of low-abundance miRNAs and the trend toward miniaturized devices has made nanomaterials significant, since they can produce a synergic effect between catalytic activity, conductivity, and biocompatibility to accelerate the signal transduction, leading to ultrasensitive detection. However, before their application for clinical diagnosis, these promising nanotechnology-based approaches must be fully validated critically for predictive sensitivity and selectivity by using patient samples. The probability of obtaining false-positive/false-negative results and impact of nanomaterials on humans and the environment should be evaluated. In these nanomaterial-based approaches, significant work needs to be devoted to the simplicity and time required for fabrication/modification, biocompatibility, and environmental friendliness. In particular, better understanding of the nanostructuring protocols will aim at guiding and tailoring the so-modified electrode platforms to meet the needs of specific applications. Furthermore, to increase the biocompatibility of nanomaterials, a significant direction to explore is the use of mild biofunctional ways for the attachment of the biomolecules on the surface of the nanostructures. Some other significant challenges involve reliable microarray fabrication, integration into automated systems (essential to achieve widespread POC use), optimization with real biological samples, and increased speed of analysis. POC testing should be fast enough to relay results quickly, leading to an improved prognosis and aiding in surgical decisions. In summary, there are significant challenges to meet before POC electrochemical arrays for cancer diagnosis through detection of miRNA expression profiles become a reality. The effort devoted by the scientific community in this research field is unprecedented, allowing a certain optimism for the years to come, in which the introduction of these miRNAs and the identification of novel ones in clinical practice seems about to become reality.
Electrochemical genosensors for cancer-related miRNAs Acknowledgments The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ1642) is gratefully acknowledged.
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