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Nanozymes and aptamer-based biosensing
Journal Pre-proofs Nanozymes and aptamer-based biosensing Bandhan Chatterjee, Soon Jyoti Das, Anjali Anand, Tarun Kumar Sharma PII: DOI: Reference:
S2589-2991(19)30105-3 https://doi.org/10.1016/j.mset.2019.08.007 MSET 115
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Materials Science for Energy Technologies
Received Date: Revised Date: Accepted Date:
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Please cite this article as: B. Chatterjee, S. Jyoti Das, A. Anand, T. Kumar Sharma, Nanozymes and aptamer-based biosensing, Materials Science for Energy Technologies (2019), doi: https://doi.org/10.1016/j.mset.2019.08.007
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Nanozymes and aptamer-based biosensing Bandhan Chatterjee, Soon Jyoti Das, Anjali Anand, Tarun Kumar Sharma* 2Multidisciplinary
Clinical and Translational Research Group, Translational Health Science and Technology Institute (THSTI), Faridabad, Haryana, India.
*Corresponding Authors *Email: *Tarun K. Sharma, [email protected]; Telephone: +91 8800931999.
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Abstract Nanozymes are a class of artificial enzymes that have dimensions in nano range. The constant development in the field of nanotechnology and material science confers the exciting possibility of exploring novel nanozymes and their possible uses. Nanozymes are increasingly finding their utility in the field of diagnostics and biosensing. The amenability of the nanozymes to work in tandem with aptamers, another relatively recently discovered superior surrogate for antibodies, has opened a completely new arena in the field of biosensing and diagnostics. The nanozymes-based aptasensors are compatible with the existing as well newly developed sensing platforms ranging from colorimetry to electrochemical. We herein concisely review the development of nanozymes and some recent advances in nanozymes-based aptasensing. Keywords: Nanozyme; aptamer, biosensing, gold nanoparticles.
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Introduction Enzymes are catalysts of biomolecular composition chiefly proteins, a few of nucleic acids[1]. They typically function in biological systems where the conditions are relatively mild when compared with industrial catalysts which function in hostile environments like high temperature and pressure, extreme pH, etc[2]. However, it is the outstanding specificity and catalytic efficiency of enzymes that entices researchers to find a way to employ enzymes in industrial uses and if not possible, then find a suitable surrogate of them capable of functioning in conditions that are not native to them. The enzymes found their application in milder industries like food, medical and biological industries, though remained plagued their inherent shortcoming as low operational stability, sensitivity to operational environments (pH, solvents, temperature, etc.), prohibitive cost of production and purification and their recycling and salvage process. Although DNA recombinant technology, protein engineering and other empirical designs have been able to address many of these issues [1,3,4] still a surrogate is needed to alleviate many of the remaining challenges. The search for materials capable of mimicking enzymes or as Ronald Breslow called them “Artificial enzymes”[5] has been parallel to theses development all these years. Last few decades of intense research on artificial enzymes have yielded some of the highly stable and economical substitutes for the natural enzymes[6]. Materials like porphyrins, metal complexes, polymers, cyclodextrins, supramolecules and some biomolecules have been extensively researched for the possibility of them mimicking like enzymes and have yielded encouraging results in this regard[7–9]. The subject has been thoroughly reviewed in numerous occasion[9–11] but is beyond the scope of current review. The prime focus of this review is to describe and discuss the recent findings in the field of nanozyme and its application in conjuction with aptamer in biosensing. 3
Nanozymes The advent of nanotechnology brought an altogether new level of possibilities which were hitherto unseen as their properties are starkly novel and different than their bulk counterparts[12,13]. With these novel properties and possible usage of nanomaterials pouring in at an unprecedented rate in the last decade[14], the speculation of finding nano dimensional artificial enzymes of novel and immensely better properties than the previous one gains momentum. Manea et al. in a pioneering paper christened the gold nanoparticles as “nanozymes, in analogy to the nomenclature of catalytic polymers (synzymes)” due to their ability to catalyze the cleavage of phosphate diesters[15]. Later on, the term assumed a sobriquet for nanomaterials exhibiting catalytic/enzymatic activities. The ambit of nanomaterials been subsumed under nanozymes ranges from simple metal and metal oxide nanoparticles, metal nanoclusters, dots (quantum and carbon both) nanotubes, nanowires and to multiple metal-organic frameworks (MOFs)[16,17]. These nanozymes exhibit excellent catalytic activities with lower cost of production, higher operational robustness and self-life, and provides greater degree of room for required modifications than the conventional enzymes[14]. For the sake of understanding nanozymes it can be broadly categorized into two big families which then can further be furcated into various subcategories. These two large families are a) oxidoreductase family, the member of this family basically involved in redox catalysis and can have catalase, superoxide dismutase, oxidase, peroxidase, and nitrate reductases, like properties (2) hydrolase family, basically involved in catalyzing split and can include phosphatase, protease, nuclease, esterase, and silicatein mimic[18]. In recent years catalytic activity of nanozymes, primarily peroxidase like activity has been utilized extensively for various biosensing applications in combination with modern molecular recognition element like aptamers. Aptamers has ability to tune peroxidase like activity of nanozymes and this combination of nanozyme and aptamer can act 4
like turn-off/turn-on switch for peroxidase like activity of nanozymes to sense various analytes. A detailed description about aptamers is stated in below mentioned section. This review is an attempt to discuss the application of nanozyme and aptamers for various biosensing applications.
Aptamers Traditionally nucleic acids are known to store genetic information but in 1990 their nonstereotypical function was discovered by two independent groups Ellington and Szostak, 1990[19]; and Tuerk and Gold, 1990[20]. They have discovered a new class of nucleic acid called ‘aptamers’. Aptamers are ssDNA, RNA or peptide molecules that can acquire a unique two- or threedimensional structure to interact with its cognate target through various physio-chemical interactions[21]. Due to their unique target-specific interactions and ease in synthesis and functionalization they have emerged as a strong chemical rival of antibody. Further, their ability to adsorb on to the various nanoparticles or nanozymes has helped the researchers to create an array of colorimetric and electrochemical assays[22–24]. Owing to these attributes over the last three decades aptamers have extensively used to develop various diagnostic or biosensing platforms to detect variety of analytes ranging from small ions[25,26], small organic molecules[27], proteins[28,29] to whole cell[30,31]. With the ever-increasing burden of infectious disease and contamination of food and water sources, there is urgent need of facile, rapid and affordable detection platforms. Although all these entities can be rapidly detected with the current prevalent techniques most of them have prerequisite of expensive and complex instrumentation, that further requires technical expertise and has to be housed in sophisticated infrastructure and simply unable to operate at onsite.
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Techniques like Polymerase chain reaction (PCR), Flow Cytometry, Enzyme-linked immunosorbent assay (ELISA), High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Mass Spectroscopy and other analytical techniques are required for the detection of the pathogens, metabolites, antibiotics, heavy metals, pesticides, and many other small molecules[22,32–34]. However, the coupling of molecular recognition ability of aptamers to inherent catalytic activity of nanozymes has provided us with an extraordinary opportunity to develop detection systems or facile sensors that work on nil or minimalistic instrumentation and skilled manpower for rapid and accurate detection [33,35–37] of various analytes. In the following section we will discuss some of the recent advances in the field of nanozyme and aptamer-based detection of entities ranging from whole cells like pathogens to small molecules of concern like antibiotics and toxins. Nanozymes-based aptasensors for biosensing Colorimetry remains one of the most popular detection platforms due to its simplicity. The minimum requirement of instruments, technical expertise and option to be visualized by naked eyes can be attributed to the success of the detection platform [38–40]. We herein list few of the recent advances in the field of colorimetric detection of analytes based on peroxidase enzyme-like activity of nanoparticles (nanozyme) and molecular recognition ability of aptamers. The utility of gold nanoparticles (GNPs) as nanozymes was quickly recognized by the research community due to its excellent ability to mimic peroxidase activity. The tendency of the GNPs to bind ssDNA in a reversible manner make them an attractive option for aptamer-based sensing. The potential of the the phenomenon was quickly regonized and was intensely researched for biosensing applications [41] . The underlying principle is that analyte-specific aptamers bind with the GNPs in a nonspecific manner, in-process layering their surface. This renders the surface of 6
GNPs inaccessible for nanozyme activity. However, in the presence of cognate target the aptamers abandon the GNPs surface to bind its target, in turn vacating the GNPs surface to resume their nanozyme activity[37,42,43]. The utility of the phenomenon was illustrated in a pioneering work by Sharma and coworkers, where they detected kanamycin with the aptamer nanozyme-based colorimetric assay. They allowed to interact GNPs with the kanamycin specific aptamers, which covered the GNPs surface. This impedes the GNPs mediated 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation, which turns colorless TMB into its blue-colored oxidized form. However, with the addition of kanamycin in the reaction mixture, the aptamers desert the GNPs surface after binding with the kanamycin, restoring the TMB oxidation by GNPs. the extent of oxidation (intensity of the blue color) essentially reflects the kanamycin concertation as more kanamycin means more amount of aptamer leaving the GNPs surface so providing more GNPs surface for TMB oxidation. They reported a limit of detection (LOD) of 1.49 nM and was able to efficiently differentiate between kanamycin from other antibiotics namely penicillin, ampicillin and streptomycin[42] Weerathunge and coworkers have used the aforementioned principal (GNPs based nanozyme assay) to develop a detection system for Human norovirus (NoV) (Figure 1). NoV is primarily a group of icosahedral, non-enveloped viruses with a positive-sense single-stranded RNA genome and is responsible for acute viral gastroenteritis worldwide, commonly known as “winter vomiting disease”. The disease is primarily communicated by person to person contact but can also be spread through consumption of contaminated food and water. The gold standards for the detection of NoV infection remain reverse transcription quantitative polymerase chain reaction (RTqPCR), though alternate detection strategies are developing. The authors employed Murine Norovirus (MNV) specific aptamer (AG3) as molecular recognition element. The authors have used MNV as a 7
surrogate for human NoV as they are easily cultivable and ideal surrogate for human NoV. The developed test was able to detect MNV in just 10 min colorimetrically with a LOD of 3 viruses in 100 µL sample (equivalent to 30 viruses/mL), that is enough to detect even infancy of the NoV infection. The performance of developed test remain uncompromised to inference from other bacteria and viruses, was able to detect NoV in human serum and shellfish homogenate[44].
Figure 1: Graphical illustration of the working principle of the aptamer and nanozyme based detection of NoV (Reprinted with permission from ref. 44. Copyright (2019) American Chemical Society.) Here the surface of gold nanoparticles are passivated with AG3 aptamers specific to norovirus thus effectively blocking the TMB oxidation. However, in the presence of norovirus the aptamers bind with them and desert the GNP surface and consequently enabling the TMB oxidation producing the blue color. The aptamers due to their high specificity towards the norovirus did not cross reacting with non-target pathogens (E.coli, S. aureus and MS2) thus continued to passivate the GNP surface and blocking the TMB oxidation, thus not producing the blue color. In a similar approach, Sun and coworkers measured Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, a common cause of contamination in agricultural products especially wheat, corn, barley, soybeans, as well as oats. Zearalenone (ZEN) causes strong mutagenicity, teratogenicity, neurotoxicity, reproductive toxicity and induces cancer. The sensing
8
relied on the ability of the GNPs to oxidize the TMB in the presence of H2O2. For this they employed citrate stabilized 13 nm monodisperse gold solution and TMB as the reporter agent, whose oxidation was measured as a function of recovery of the nanozyme activity. This, in turn, was a measure of ZEN in the detection medium. The aptasensor was able to detect up to 10 ng·mL−1 and maintained linearity in the range of 10-250 ng·mL−1 but was moderately immune to interference by AFB1, OTA and some common metal ions (Ca2+, Na+, Mg2+, Zn2+). The detection test was able to sense ZEN in corn and corn oils with results that are comparable with ELISA. [45] Ouyang and coworkers exploited the nanozyme nature of GNPs in a noble manner to detect lead (Pb 2+) ions in water. They relied on the ability of the GNPs to couple the reduction of Au 3+ to Au 0
and oxidation of H2O2. Both Au 3+ and H2O2 were adsorbed on the surface of GNPs, where the
free surface electrons of the GNPs accentuates the redox electron transfer to form H2O2 to Au 3+ reducing it to first Au+ and Au0 forming the GNPs which are detected by Surface-enhanced Raman spectroscopy (SERS) using Victoria Blue B (VBB) as molecular probes (Figure 2). The SERS peak at 1614 cm−1 intensity increase with an increase in synthesis of more GNPs. Interestingly, this conversion is impeded with the addition of aptamers, which binds with the GNPs and blocks the redox electron transfer, hence lessens the SERS signal. However, with the addition of Pb
2+
into the medium the aptamers desert the GNPs surface, and the redox reaction resumes. This leads to accentuation of the SERS signal. The ability of the GNPs to conduct this redox reaction is greatly enhanced with the reduction in size of GNPs, as the smaller GNPs offer larger surface area for the redox reaction. The authors investigated four different sized GNPs for it, viz 10 nm, 30 nm, 60nm and 95 nm, and they found that the strongest SERS signal was produced with 10 nm GNPs, which gradually diminishes with increase in nanoparticle size, with 95 nm-sized GNPs depicting very weak catalytic activity. The 10 nm GNPs displayed strong catalytic activity, excellent linearity in 9
the range of 0.13–53.33 nmol/L with a LOD of 0.07 nmol/L. The test was minimally influenced by the presence of other ions, marking the selectivity of it. The test able to detect Pb
2+
in real
samples like pond water.[46]
Figure 2 Schematic representation of the a) Principle of AuNP catalysis of HAuCl4-H2O2, here the GNPs surface electron facilitates the conversion of Au 3+ into gold nanoparticle (Au0) with the help of H2O2 b) AuNP catalysis-SERS detection of Pb2+ (Reprinted with permission from ref. 46. Copyright 2017, Elsevier). With the layering of GNPs surface with aptamers the reducition of Au 3+ into
Au0 is blocked thus the SERS signal intensity declines, however, with the addition of Pb2+
the aptamers deserts the GNP surface after binding with the Pb2+, as a result enabling the reduction process again and thus increasing the SERS signal.
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Huang and coworkers reported the spinel-type manganese cation substituted cobalt oxide (MnCo2O4) submicrospheres depicts oxidase-like activity, and the catalytic activity is superior to most of the reported mono-metal oxides (Co3O4, Mn3O4, MnO2, and CeO2, etc.) and bimetallic oxides (MnFe2O4). The higher catalytic efficiency can be attributed to the synergistic effect between Mn and Co in the spinel structure. The oxidase-like activity can be ascribed to the fastredox cycles (Co3+/Co2+ and Mn3+/Mn2+) happening at the surface of the spheres. Further, MnCo2O4 binds with single-stranded DNA in a reversible manner and the affinity of the sequence towards MnCO2O4 submicrospheres is dictated by the sequence’s base composition. The authors illustrated the amenability of this nanozyme for aptamer-based biosensing, by detecting ochratoxin A (OTA), one of the most widespread mycotoxins. The OTA is reported to cause strong hepatotoxicity, renal toxicity, teratogenic, carcinogenic, and also have mutagenic effect. Further, it has high chemical and thermal stability and is difficult to remove once it enters the food chain. The sensing approach remains similar as in many nanozyme-based colorimetric approaches, OTA specific aptamer binds on the surface of the MnCO2O4 submicrospheres and blocking the electron transfer between MnCO2O4 and TMB. However, with the addition of OTA in the medium the aptamers leave the MnCo2O4 surface to bind OTA, enabling the oxidation of TMB. The event is measurable with colorimetry the test displayed linearity in 0.1–10 ng/mL range with a LOD of 0.08 ng/mL which is better than many currently used techniques like ELISA and HPLC[47]. The aptasensor also displayed an impressive specificity for OTA, as it charted an intense response for OTA at 3 ng/mL, however did not produce any cognizable response even at 30 ng/mL of OTA coexisting analogs such as ochratoxin B, ochratoxin C, ochratoxin B, zearalenone, and aflatoxin B1. Further, the detection of OTA remains unaffected in presence of aforementioned analogs..
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Tian and coworkers reported another aptasensor for colorimetric detection of OTA by using a nanozyme-based cascade reaction (MnO2 nanosheets, as nanozyme and alkaline phosphatase ALP for signal amplification). They immobilized biotin-labeled OTA specific aptamers onto the streptavidin magnetic beads (MBs). These were then subjected to a biotin-labeled primer which is complementary to partial length of aptamer’s free end and forms a partial ds DNA structure having free biotin label- at one end and aptamers anchored to MBs on other ends. Following this alkaline phosphatase streptavidin (SA-ALP) is introduced in the medium, which attaches to the biotinlabeled end of the primers and finally forming DNA-ALP-immobilized MBs. When OTA is introduced into the reaction medium its presence induces a structural change in the aptamer that is detrimental to the partial ds DNA structure, forcing the primer-ALP to detach from the aptamer MBs structure. The aptamer anchored MBs are removed from the reaction medium by magnetic separation and ascorbic acid-2-phosphate (AAP) is introduced into the medium. This initiates the cascading reaction first the AAP is hydrolyzed to ascorbic acid (AA) by the remaining
ALP
attached to the primers. The produced AA due to its redox capability then reduces MnO2 nanosheets to Mn2+ ions. This fetters the oxidase-like activity of the MnO2 nanosheets and which in turn can no longer oxidize TMB into a colored product. However, in the absence of OTA this all cascading events is blocked so the nanosheets oxidize the TMB into a colored product (Figure 3). The sensor designed was able to produce a linear response for OTA in the range of 1.25-250 nM with a LOD of 0.069 nM. The aptasensor was able to discriminate between OTA and its structural analogs such as ochratoxin B (OTB), aflatoxin B1 (AFB1), citrinin, zearalenone (ZEN), altenuene (ALT), and tenuazonic acid (TeA) and detect OTA in grape juice.[27]
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Figure:3 Schematic representation of the nanozyme-based cascade colorimetric aptasensor for OTA detection (Reprinted with permission from ref. 27. Copyright 2019, Royal society of chemistry). The nanozyme based catalysis can also be used in tandem with electrochemical platforms, for signal generation, and this provides an additional advantage of being highly sensitive and requires very small amount of analytes to be tested [48–50]. Das and coworkers have used the strategy to detect Pseudomonas aeruginosa (PA), an infectious bacterial agent that causes widespread contamination of water and food particularly in Asia, Europe and America[51]. The bacteria are also known to cause many nosocomial infections[51] and infecting immunocompromised patients[52]. The authors used a PA specific aptamer as a molecular probe and GNPs as nanozyme. Presence of aptamer passivate the GNPs surface and block the nanozyme activity of nanoparticles thus prevent the oxidation of TMB. They interacted the GNPs with the aptamers rendering the GNPs surface covered and thus unable to oxidize the TMB. However, in the presence of PA, the aptamers leave the GNPs surface and oxidation of TMB resumes. The oxidation of TMB can be stopped with H2SO4. The presence of acid (H2SO4) convert TMB to its electrochemically active form and this form was employed to assess the amperometric response of sensor in presence of PA [50] (Figure 4).
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Figure:4 Principal of aptamer-nanozyme based colorimetric and electrochemical assay for the detection of Pseudomonas aeruginosa (PA). (Reprinted with permission from ref. 50. Copyright 2019, springer). Here, after the nanozyme reaction is complete the reaction is blocked with H2SO4, and the response is measure with the amperometry on a screen prnted electrode. The same strategy is used by Wang and coworkers to detect kanamycin[53], with a slight deviation as they used thionine instead of TMB as a reporter agent. They interacted kanamycin specific aptamers with tyrosine-capped GNPs, which then blocks the oxidation of thionine by H2O2. With the introduction of kanamycin in the medium, the aptamer deserts the GNPs surface to catalyze the thionine oxidation by H2O2, which can be measured with differential pulse voltammetry (DPV), on the surface of gold electrode (Figure 5 ). The method displayed linearity in the detection range of 0.1-60 nM and a LOD of 0.06 nM. As the electrodes were not modified for the measurement purposes, they can be simply washed with ethyl alcohol and ultrapure water and reused. Interestingly the detection system was able to recognize kanamycin B, however, displayed negligible response with other aminoglycoside antibiotics (streptomycin, gentamicin and neomycin) and non-aminoglycoside antibiotics (tetracycline). To gauge the utility of the sensing system the authors successfully measure upto 0.73 nM of kanamycin in honey samples.
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Figure 5: Working principle for the proposed assay for detection of kanamycin (Reprinted with permission from ref. 53. Copyright 2016, Elsevier). Zhao and coworkers developed a nanozyme based electrochemical detection of Mucin 1. Mucin is a family of heavily glycosylated proteins produced by epithelial cells. Mucin 1 (MUC1) overexpression is associated with cancer and is increasingly gaining attention as a reliable biomarker for cancer detection. To detect MUC1 authors have relied on catalytic hairpin assembly (CHA) for the hundreds-fold catalytic amplification reaction. They employed three hairpin DNA (Apt-HP1, HP2 and HP3) to fabricate the sensor on a gold electrode. First the Apt-HP1 was reacted with the sample containing MUC1 and forming MUC1-aptamer binding complex (MUC1-A), and in-process exposes a sequence on Apt-HP1 which is complementary to a minor region of APT HP2, already immobilized on the gold electrode surface. After this platinum-palladium nanoparticles (PtPdNPs) tagged APT HP3 is added to the electrode surface, which is complementary to a section of APT HP2 (other than region complementary to Apt-HP1). Consequentially, MUC1-A is dislodged from the APT HP 2 via the strand displacement process, 15
leaving free MUC1-A for binding with another free APT HP 2 and initiating another CHA. Here the reporter probe is PtPdNPs, which due to virtue of its mimicking peroxidase, acts as a nanozyme capable of oxidizing TMB with H2O2 and generate redox electrochemical response measured with cyclic voltammetry (Figure 6). The detector displayed detection linearity in the range of 100 fg mL−1 to 1 ng mL−1and revealed a LOD of 16 fg mL−1. The developed sensor was able to detect MUC1 in human serum samples, although minor inference was observed with higher human serum albumin (HSA) concentration in vitro, thus for application with human serum, the serum needs to be diluted a few folds to reduce the inference from HSA[28].
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Figure 6: Working principle of the aptasensor for the detection of MUC1 (Reprinted with permission from ref. 28. Copyright 2019, Elsevier). Ou and coworkers developed a sensor based on DNA nanostructures and flower-like nanozymes for detection of Human Epidermal Growth Factor Receptor 2 (HER2), a protein biomarker for breast cancer detection. The biomarkers like Human Epidermal Growth Factor Receptor 2 (HER2), Vascular Endothelial Growth Factor (VEGF), Mucin 1 (MUC1) and Carcinoembryonic Antigen (CEA) characteristically associated with breast cancer are typically diagnosed with antibody-based immunochemistry or ELISA. However, the inherent averse of the antibodies for need-based modifications, besides their inherent limitation exhorts for exploration of alternate options. The authors used aptamers in an ingenious manner to detect the HER2 with detection linearity in the range of 0.1–100.0 ng/mL and a LOD of 0.08. ng/mL Further, in this study the authors also address the issue related to random entanglement and aggregation when not anchored properly and thus hindering their proper binding with the analyte. The authors anchored the aptamer on a Tetrahedral DNA nanostructure (TDN), formed by hybridization of four ss DNA sequences (aptamer being a part of the fourth sequence itself forming a pendent like structure) which themselves are thiolated at 3 ends. These thiolated ends cement the TDN-aptamer firmly on the gold electrode via thiolgold interaction. The recognition of HER2 is carried out in two steps, first the anchored aptamer recognizing the HER2 and capturing it on the electrode surface (as the whole apt-TDN, is anchored on electrode vis Au-S bond). In a separate step another aptamer recognizing a different epitope of HER2, and HRP are loaded on a Mn3O4/Pd@Pt nanocarrier to form nanoprobe 1. This nanoprobe 1 then binds with the already captured HER2 on electrode surface. To further have an accentuated signal amplification, a cDNA (complementary to sequence in nanopoprobe 1) is tagged with Pd@Pt/HRP, nanoprobe 2 and is added to the surface which joins the already formed complex with DNA hybridization with nanoprobe 1. This structure now has three nanozymes working in 17
tandem for signal generation and amplification viz. the Mn3O4 nanoflowers, Pd@Pt nanoflowers and HRP, by catalyzing the oxidation of hydroquinone (HQ) with H2O2 to benzoquinone (BQ) (Figure 7). The response in recorded by differential pulse voltammetry (DPV) for more sensitive detection. The test was able to perform in serum samples with a error within 10% when compared with standard ELISA, marking the potential of the biosensors for clinical diagnosis [54].
Figure 7: Principal of the electrochemical dual-aptamer biosensor for HER2 detection (HRP: horseradish peroxidase; GE: gold electrode; TDN: tetrahedral DNA nanostructure; MCH: 6mercapto-1-hexanol; HQ: hydroquinone; BQ: benzoquinone; DPV: differential pulse voltammetry). (Reprinted with permission from ref. 54. Copyright 2019, Royal society of chemistry).
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Table 1: A A comprehensive list of nanozyme and aptamer based biosensors and their attributes. S.NO.
DETECTION MARKER
DETECTION TECHNIQUE
1.
Human norovirus (NoV) Zearalenone (ZEN) Lead (Pb2+) ions
colorimetry
Ochratoxin A (OTA) Ochratoxin A (OTA)
colorimetry
Pseudomonas aeruginosa
electrochemical
2. 3.
4. 5.
6.
colorimetry Surfaceenhanced Raman spectroscopy (SERS)
colorimetry
DETECTION RANGE 20-3,300 norovirus/mL
10-250 ng·mL−1 0.13–53.33 nmol/L
LIMIT OF DETECTION
REFERENCE
3 viruses in 100 µL 10 ng·mL−1
[44]
0.07 nmol/L
[46]
[45]
0.1–10 0.08 ng/mL ng/mL 1.25-250 nM 0.069 nM
[47]
60 CFU/mL
[50]
[27]
19
7.
kanamycin
electrochemical 0.1-60 nM
0.06 nM
[53]
8.
Mucin 1 (MUC1)
16 fg mL−1
[55]
9.
Human Epidermal Growth FactorReceptor 2 (HER2)
electrochemical 100 fg mL−1 to 1 ng mL−1 electrochemical 0.1–100.0 ng/mL
0.08ng/mL
[54]
Conclusions and future directions In summary, in the current review, we have given a brief overview of the intrinsic peroxidase-like activity of nanoparticles (nanozymes) and also through a light on application of nanozymes in combination with aptamers for biosensing applications. The aptamer-nanozyme system has wider utility and has shown its value to detect variety of analytes ranging from small molecules to whole cell. Further, adaptation of this system on to an electrochemical sensing platform has further increased the sensitivity and low-end detection limit. The aforementioned advances in this field have given a strong foundation to develop robust, rapid, sensitive and cost-effective point-of-care
20
device similar to lateral flow in near future that can be used as a Target Product Profile 1 (TPP-1) diagnostics[56]. Acknowledgments T.K.S. wishes to thank the Department of Biotechnology, Govt. of India for DBT-Innovative Young Biotechnologist Award (BT/010/IYBA/2016/10). T.K.S. also thanks Translational Health Science and Technology Institute (THSTI) for providing core grant funds to support.
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S2589-2991(19)30105-3 https://doi.org/10.1016/j.mset.2019.08.007 MSET 115
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Please cite this article as: B. Chatterjee, S. Jyoti Das, A. Anand, T. Kumar Sharma, Nanozymes and aptamer-based biosensing, Materials Science for Energy Technologies (2019), doi: https://doi.org/10.1016/j.mset.2019.08.007
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Nanozymes and aptamer-based biosensing Bandhan Chatterjee, Soon Jyoti Das, Anjali Anand, Tarun Kumar Sharma* 2Multidisciplinary
Clinical and Translational Research Group, Translational Health Science and Technology Institute (THSTI), Faridabad, Haryana, India.
*Corresponding Authors *Email: *Tarun K. Sharma, [email protected]; Telephone: +91 8800931999.
1
Abstract Nanozymes are a class of artificial enzymes that have dimensions in nano range. The constant development in the field of nanotechnology and material science confers the exciting possibility of exploring novel nanozymes and their possible uses. Nanozymes are increasingly finding their utility in the field of diagnostics and biosensing. The amenability of the nanozymes to work in tandem with aptamers, another relatively recently discovered superior surrogate for antibodies, has opened a completely new arena in the field of biosensing and diagnostics. The nanozymes-based aptasensors are compatible with the existing as well newly developed sensing platforms ranging from colorimetry to electrochemical. We herein concisely review the development of nanozymes and some recent advances in nanozymes-based aptasensing. Keywords: Nanozyme; aptamer, biosensing, gold nanoparticles.
2
Introduction Enzymes are catalysts of biomolecular composition chiefly proteins, a few of nucleic acids[1]. They typically function in biological systems where the conditions are relatively mild when compared with industrial catalysts which function in hostile environments like high temperature and pressure, extreme pH, etc[2]. However, it is the outstanding specificity and catalytic efficiency of enzymes that entices researchers to find a way to employ enzymes in industrial uses and if not possible, then find a suitable surrogate of them capable of functioning in conditions that are not native to them. The enzymes found their application in milder industries like food, medical and biological industries, though remained plagued their inherent shortcoming as low operational stability, sensitivity to operational environments (pH, solvents, temperature, etc.), prohibitive cost of production and purification and their recycling and salvage process. Although DNA recombinant technology, protein engineering and other empirical designs have been able to address many of these issues [1,3,4] still a surrogate is needed to alleviate many of the remaining challenges. The search for materials capable of mimicking enzymes or as Ronald Breslow called them “Artificial enzymes”[5] has been parallel to theses development all these years. Last few decades of intense research on artificial enzymes have yielded some of the highly stable and economical substitutes for the natural enzymes[6]. Materials like porphyrins, metal complexes, polymers, cyclodextrins, supramolecules and some biomolecules have been extensively researched for the possibility of them mimicking like enzymes and have yielded encouraging results in this regard[7–9]. The subject has been thoroughly reviewed in numerous occasion[9–11] but is beyond the scope of current review. The prime focus of this review is to describe and discuss the recent findings in the field of nanozyme and its application in conjuction with aptamer in biosensing. 3
Nanozymes The advent of nanotechnology brought an altogether new level of possibilities which were hitherto unseen as their properties are starkly novel and different than their bulk counterparts[12,13]. With these novel properties and possible usage of nanomaterials pouring in at an unprecedented rate in the last decade[14], the speculation of finding nano dimensional artificial enzymes of novel and immensely better properties than the previous one gains momentum. Manea et al. in a pioneering paper christened the gold nanoparticles as “nanozymes, in analogy to the nomenclature of catalytic polymers (synzymes)” due to their ability to catalyze the cleavage of phosphate diesters[15]. Later on, the term assumed a sobriquet for nanomaterials exhibiting catalytic/enzymatic activities. The ambit of nanomaterials been subsumed under nanozymes ranges from simple metal and metal oxide nanoparticles, metal nanoclusters, dots (quantum and carbon both) nanotubes, nanowires and to multiple metal-organic frameworks (MOFs)[16,17]. These nanozymes exhibit excellent catalytic activities with lower cost of production, higher operational robustness and self-life, and provides greater degree of room for required modifications than the conventional enzymes[14]. For the sake of understanding nanozymes it can be broadly categorized into two big families which then can further be furcated into various subcategories. These two large families are a) oxidoreductase family, the member of this family basically involved in redox catalysis and can have catalase, superoxide dismutase, oxidase, peroxidase, and nitrate reductases, like properties (2) hydrolase family, basically involved in catalyzing split and can include phosphatase, protease, nuclease, esterase, and silicatein mimic[18]. In recent years catalytic activity of nanozymes, primarily peroxidase like activity has been utilized extensively for various biosensing applications in combination with modern molecular recognition element like aptamers. Aptamers has ability to tune peroxidase like activity of nanozymes and this combination of nanozyme and aptamer can act 4
like turn-off/turn-on switch for peroxidase like activity of nanozymes to sense various analytes. A detailed description about aptamers is stated in below mentioned section. This review is an attempt to discuss the application of nanozyme and aptamers for various biosensing applications.
Aptamers Traditionally nucleic acids are known to store genetic information but in 1990 their nonstereotypical function was discovered by two independent groups Ellington and Szostak, 1990[19]; and Tuerk and Gold, 1990[20]. They have discovered a new class of nucleic acid called ‘aptamers’. Aptamers are ssDNA, RNA or peptide molecules that can acquire a unique two- or threedimensional structure to interact with its cognate target through various physio-chemical interactions[21]. Due to their unique target-specific interactions and ease in synthesis and functionalization they have emerged as a strong chemical rival of antibody. Further, their ability to adsorb on to the various nanoparticles or nanozymes has helped the researchers to create an array of colorimetric and electrochemical assays[22–24]. Owing to these attributes over the last three decades aptamers have extensively used to develop various diagnostic or biosensing platforms to detect variety of analytes ranging from small ions[25,26], small organic molecules[27], proteins[28,29] to whole cell[30,31]. With the ever-increasing burden of infectious disease and contamination of food and water sources, there is urgent need of facile, rapid and affordable detection platforms. Although all these entities can be rapidly detected with the current prevalent techniques most of them have prerequisite of expensive and complex instrumentation, that further requires technical expertise and has to be housed in sophisticated infrastructure and simply unable to operate at onsite.
5
Techniques like Polymerase chain reaction (PCR), Flow Cytometry, Enzyme-linked immunosorbent assay (ELISA), High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Mass Spectroscopy and other analytical techniques are required for the detection of the pathogens, metabolites, antibiotics, heavy metals, pesticides, and many other small molecules[22,32–34]. However, the coupling of molecular recognition ability of aptamers to inherent catalytic activity of nanozymes has provided us with an extraordinary opportunity to develop detection systems or facile sensors that work on nil or minimalistic instrumentation and skilled manpower for rapid and accurate detection [33,35–37] of various analytes. In the following section we will discuss some of the recent advances in the field of nanozyme and aptamer-based detection of entities ranging from whole cells like pathogens to small molecules of concern like antibiotics and toxins. Nanozymes-based aptasensors for biosensing Colorimetry remains one of the most popular detection platforms due to its simplicity. The minimum requirement of instruments, technical expertise and option to be visualized by naked eyes can be attributed to the success of the detection platform [38–40]. We herein list few of the recent advances in the field of colorimetric detection of analytes based on peroxidase enzyme-like activity of nanoparticles (nanozyme) and molecular recognition ability of aptamers. The utility of gold nanoparticles (GNPs) as nanozymes was quickly recognized by the research community due to its excellent ability to mimic peroxidase activity. The tendency of the GNPs to bind ssDNA in a reversible manner make them an attractive option for aptamer-based sensing. The potential of the the phenomenon was quickly regonized and was intensely researched for biosensing applications [41] . The underlying principle is that analyte-specific aptamers bind with the GNPs in a nonspecific manner, in-process layering their surface. This renders the surface of 6
GNPs inaccessible for nanozyme activity. However, in the presence of cognate target the aptamers abandon the GNPs surface to bind its target, in turn vacating the GNPs surface to resume their nanozyme activity[37,42,43]. The utility of the phenomenon was illustrated in a pioneering work by Sharma and coworkers, where they detected kanamycin with the aptamer nanozyme-based colorimetric assay. They allowed to interact GNPs with the kanamycin specific aptamers, which covered the GNPs surface. This impedes the GNPs mediated 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation, which turns colorless TMB into its blue-colored oxidized form. However, with the addition of kanamycin in the reaction mixture, the aptamers desert the GNPs surface after binding with the kanamycin, restoring the TMB oxidation by GNPs. the extent of oxidation (intensity of the blue color) essentially reflects the kanamycin concertation as more kanamycin means more amount of aptamer leaving the GNPs surface so providing more GNPs surface for TMB oxidation. They reported a limit of detection (LOD) of 1.49 nM and was able to efficiently differentiate between kanamycin from other antibiotics namely penicillin, ampicillin and streptomycin[42] Weerathunge and coworkers have used the aforementioned principal (GNPs based nanozyme assay) to develop a detection system for Human norovirus (NoV) (Figure 1). NoV is primarily a group of icosahedral, non-enveloped viruses with a positive-sense single-stranded RNA genome and is responsible for acute viral gastroenteritis worldwide, commonly known as “winter vomiting disease”. The disease is primarily communicated by person to person contact but can also be spread through consumption of contaminated food and water. The gold standards for the detection of NoV infection remain reverse transcription quantitative polymerase chain reaction (RTqPCR), though alternate detection strategies are developing. The authors employed Murine Norovirus (MNV) specific aptamer (AG3) as molecular recognition element. The authors have used MNV as a 7
surrogate for human NoV as they are easily cultivable and ideal surrogate for human NoV. The developed test was able to detect MNV in just 10 min colorimetrically with a LOD of 3 viruses in 100 µL sample (equivalent to 30 viruses/mL), that is enough to detect even infancy of the NoV infection. The performance of developed test remain uncompromised to inference from other bacteria and viruses, was able to detect NoV in human serum and shellfish homogenate[44].
Figure 1: Graphical illustration of the working principle of the aptamer and nanozyme based detection of NoV (Reprinted with permission from ref. 44. Copyright (2019) American Chemical Society.) Here the surface of gold nanoparticles are passivated with AG3 aptamers specific to norovirus thus effectively blocking the TMB oxidation. However, in the presence of norovirus the aptamers bind with them and desert the GNP surface and consequently enabling the TMB oxidation producing the blue color. The aptamers due to their high specificity towards the norovirus did not cross reacting with non-target pathogens (E.coli, S. aureus and MS2) thus continued to passivate the GNP surface and blocking the TMB oxidation, thus not producing the blue color. In a similar approach, Sun and coworkers measured Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, a common cause of contamination in agricultural products especially wheat, corn, barley, soybeans, as well as oats. Zearalenone (ZEN) causes strong mutagenicity, teratogenicity, neurotoxicity, reproductive toxicity and induces cancer. The sensing
8
relied on the ability of the GNPs to oxidize the TMB in the presence of H2O2. For this they employed citrate stabilized 13 nm monodisperse gold solution and TMB as the reporter agent, whose oxidation was measured as a function of recovery of the nanozyme activity. This, in turn, was a measure of ZEN in the detection medium. The aptasensor was able to detect up to 10 ng·mL−1 and maintained linearity in the range of 10-250 ng·mL−1 but was moderately immune to interference by AFB1, OTA and some common metal ions (Ca2+, Na+, Mg2+, Zn2+). The detection test was able to sense ZEN in corn and corn oils with results that are comparable with ELISA. [45] Ouyang and coworkers exploited the nanozyme nature of GNPs in a noble manner to detect lead (Pb 2+) ions in water. They relied on the ability of the GNPs to couple the reduction of Au 3+ to Au 0
and oxidation of H2O2. Both Au 3+ and H2O2 were adsorbed on the surface of GNPs, where the
free surface electrons of the GNPs accentuates the redox electron transfer to form H2O2 to Au 3+ reducing it to first Au+ and Au0 forming the GNPs which are detected by Surface-enhanced Raman spectroscopy (SERS) using Victoria Blue B (VBB) as molecular probes (Figure 2). The SERS peak at 1614 cm−1 intensity increase with an increase in synthesis of more GNPs. Interestingly, this conversion is impeded with the addition of aptamers, which binds with the GNPs and blocks the redox electron transfer, hence lessens the SERS signal. However, with the addition of Pb
2+
into the medium the aptamers desert the GNPs surface, and the redox reaction resumes. This leads to accentuation of the SERS signal. The ability of the GNPs to conduct this redox reaction is greatly enhanced with the reduction in size of GNPs, as the smaller GNPs offer larger surface area for the redox reaction. The authors investigated four different sized GNPs for it, viz 10 nm, 30 nm, 60nm and 95 nm, and they found that the strongest SERS signal was produced with 10 nm GNPs, which gradually diminishes with increase in nanoparticle size, with 95 nm-sized GNPs depicting very weak catalytic activity. The 10 nm GNPs displayed strong catalytic activity, excellent linearity in 9
the range of 0.13–53.33 nmol/L with a LOD of 0.07 nmol/L. The test was minimally influenced by the presence of other ions, marking the selectivity of it. The test able to detect Pb
2+
in real
samples like pond water.[46]
Figure 2 Schematic representation of the a) Principle of AuNP catalysis of HAuCl4-H2O2, here the GNPs surface electron facilitates the conversion of Au 3+ into gold nanoparticle (Au0) with the help of H2O2 b) AuNP catalysis-SERS detection of Pb2+ (Reprinted with permission from ref. 46. Copyright 2017, Elsevier). With the layering of GNPs surface with aptamers the reducition of Au 3+ into
Au0 is blocked thus the SERS signal intensity declines, however, with the addition of Pb2+
the aptamers deserts the GNP surface after binding with the Pb2+, as a result enabling the reduction process again and thus increasing the SERS signal.
10
Huang and coworkers reported the spinel-type manganese cation substituted cobalt oxide (MnCo2O4) submicrospheres depicts oxidase-like activity, and the catalytic activity is superior to most of the reported mono-metal oxides (Co3O4, Mn3O4, MnO2, and CeO2, etc.) and bimetallic oxides (MnFe2O4). The higher catalytic efficiency can be attributed to the synergistic effect between Mn and Co in the spinel structure. The oxidase-like activity can be ascribed to the fastredox cycles (Co3+/Co2+ and Mn3+/Mn2+) happening at the surface of the spheres. Further, MnCo2O4 binds with single-stranded DNA in a reversible manner and the affinity of the sequence towards MnCO2O4 submicrospheres is dictated by the sequence’s base composition. The authors illustrated the amenability of this nanozyme for aptamer-based biosensing, by detecting ochratoxin A (OTA), one of the most widespread mycotoxins. The OTA is reported to cause strong hepatotoxicity, renal toxicity, teratogenic, carcinogenic, and also have mutagenic effect. Further, it has high chemical and thermal stability and is difficult to remove once it enters the food chain. The sensing approach remains similar as in many nanozyme-based colorimetric approaches, OTA specific aptamer binds on the surface of the MnCO2O4 submicrospheres and blocking the electron transfer between MnCO2O4 and TMB. However, with the addition of OTA in the medium the aptamers leave the MnCo2O4 surface to bind OTA, enabling the oxidation of TMB. The event is measurable with colorimetry the test displayed linearity in 0.1–10 ng/mL range with a LOD of 0.08 ng/mL which is better than many currently used techniques like ELISA and HPLC[47]. The aptasensor also displayed an impressive specificity for OTA, as it charted an intense response for OTA at 3 ng/mL, however did not produce any cognizable response even at 30 ng/mL of OTA coexisting analogs such as ochratoxin B, ochratoxin C, ochratoxin B, zearalenone, and aflatoxin B1. Further, the detection of OTA remains unaffected in presence of aforementioned analogs..
11
Tian and coworkers reported another aptasensor for colorimetric detection of OTA by using a nanozyme-based cascade reaction (MnO2 nanosheets, as nanozyme and alkaline phosphatase ALP for signal amplification). They immobilized biotin-labeled OTA specific aptamers onto the streptavidin magnetic beads (MBs). These were then subjected to a biotin-labeled primer which is complementary to partial length of aptamer’s free end and forms a partial ds DNA structure having free biotin label- at one end and aptamers anchored to MBs on other ends. Following this alkaline phosphatase streptavidin (SA-ALP) is introduced in the medium, which attaches to the biotinlabeled end of the primers and finally forming DNA-ALP-immobilized MBs. When OTA is introduced into the reaction medium its presence induces a structural change in the aptamer that is detrimental to the partial ds DNA structure, forcing the primer-ALP to detach from the aptamer MBs structure. The aptamer anchored MBs are removed from the reaction medium by magnetic separation and ascorbic acid-2-phosphate (AAP) is introduced into the medium. This initiates the cascading reaction first the AAP is hydrolyzed to ascorbic acid (AA) by the remaining
ALP
attached to the primers. The produced AA due to its redox capability then reduces MnO2 nanosheets to Mn2+ ions. This fetters the oxidase-like activity of the MnO2 nanosheets and which in turn can no longer oxidize TMB into a colored product. However, in the absence of OTA this all cascading events is blocked so the nanosheets oxidize the TMB into a colored product (Figure 3). The sensor designed was able to produce a linear response for OTA in the range of 1.25-250 nM with a LOD of 0.069 nM. The aptasensor was able to discriminate between OTA and its structural analogs such as ochratoxin B (OTB), aflatoxin B1 (AFB1), citrinin, zearalenone (ZEN), altenuene (ALT), and tenuazonic acid (TeA) and detect OTA in grape juice.[27]
12
Figure:3 Schematic representation of the nanozyme-based cascade colorimetric aptasensor for OTA detection (Reprinted with permission from ref. 27. Copyright 2019, Royal society of chemistry). The nanozyme based catalysis can also be used in tandem with electrochemical platforms, for signal generation, and this provides an additional advantage of being highly sensitive and requires very small amount of analytes to be tested [48–50]. Das and coworkers have used the strategy to detect Pseudomonas aeruginosa (PA), an infectious bacterial agent that causes widespread contamination of water and food particularly in Asia, Europe and America[51]. The bacteria are also known to cause many nosocomial infections[51] and infecting immunocompromised patients[52]. The authors used a PA specific aptamer as a molecular probe and GNPs as nanozyme. Presence of aptamer passivate the GNPs surface and block the nanozyme activity of nanoparticles thus prevent the oxidation of TMB. They interacted the GNPs with the aptamers rendering the GNPs surface covered and thus unable to oxidize the TMB. However, in the presence of PA, the aptamers leave the GNPs surface and oxidation of TMB resumes. The oxidation of TMB can be stopped with H2SO4. The presence of acid (H2SO4) convert TMB to its electrochemically active form and this form was employed to assess the amperometric response of sensor in presence of PA [50] (Figure 4).
13
Figure:4 Principal of aptamer-nanozyme based colorimetric and electrochemical assay for the detection of Pseudomonas aeruginosa (PA). (Reprinted with permission from ref. 50. Copyright 2019, springer). Here, after the nanozyme reaction is complete the reaction is blocked with H2SO4, and the response is measure with the amperometry on a screen prnted electrode. The same strategy is used by Wang and coworkers to detect kanamycin[53], with a slight deviation as they used thionine instead of TMB as a reporter agent. They interacted kanamycin specific aptamers with tyrosine-capped GNPs, which then blocks the oxidation of thionine by H2O2. With the introduction of kanamycin in the medium, the aptamer deserts the GNPs surface to catalyze the thionine oxidation by H2O2, which can be measured with differential pulse voltammetry (DPV), on the surface of gold electrode (Figure 5 ). The method displayed linearity in the detection range of 0.1-60 nM and a LOD of 0.06 nM. As the electrodes were not modified for the measurement purposes, they can be simply washed with ethyl alcohol and ultrapure water and reused. Interestingly the detection system was able to recognize kanamycin B, however, displayed negligible response with other aminoglycoside antibiotics (streptomycin, gentamicin and neomycin) and non-aminoglycoside antibiotics (tetracycline). To gauge the utility of the sensing system the authors successfully measure upto 0.73 nM of kanamycin in honey samples.
14
Figure 5: Working principle for the proposed assay for detection of kanamycin (Reprinted with permission from ref. 53. Copyright 2016, Elsevier). Zhao and coworkers developed a nanozyme based electrochemical detection of Mucin 1. Mucin is a family of heavily glycosylated proteins produced by epithelial cells. Mucin 1 (MUC1) overexpression is associated with cancer and is increasingly gaining attention as a reliable biomarker for cancer detection. To detect MUC1 authors have relied on catalytic hairpin assembly (CHA) for the hundreds-fold catalytic amplification reaction. They employed three hairpin DNA (Apt-HP1, HP2 and HP3) to fabricate the sensor on a gold electrode. First the Apt-HP1 was reacted with the sample containing MUC1 and forming MUC1-aptamer binding complex (MUC1-A), and in-process exposes a sequence on Apt-HP1 which is complementary to a minor region of APT HP2, already immobilized on the gold electrode surface. After this platinum-palladium nanoparticles (PtPdNPs) tagged APT HP3 is added to the electrode surface, which is complementary to a section of APT HP2 (other than region complementary to Apt-HP1). Consequentially, MUC1-A is dislodged from the APT HP 2 via the strand displacement process, 15
leaving free MUC1-A for binding with another free APT HP 2 and initiating another CHA. Here the reporter probe is PtPdNPs, which due to virtue of its mimicking peroxidase, acts as a nanozyme capable of oxidizing TMB with H2O2 and generate redox electrochemical response measured with cyclic voltammetry (Figure 6). The detector displayed detection linearity in the range of 100 fg mL−1 to 1 ng mL−1and revealed a LOD of 16 fg mL−1. The developed sensor was able to detect MUC1 in human serum samples, although minor inference was observed with higher human serum albumin (HSA) concentration in vitro, thus for application with human serum, the serum needs to be diluted a few folds to reduce the inference from HSA[28].
16
Figure 6: Working principle of the aptasensor for the detection of MUC1 (Reprinted with permission from ref. 28. Copyright 2019, Elsevier). Ou and coworkers developed a sensor based on DNA nanostructures and flower-like nanozymes for detection of Human Epidermal Growth Factor Receptor 2 (HER2), a protein biomarker for breast cancer detection. The biomarkers like Human Epidermal Growth Factor Receptor 2 (HER2), Vascular Endothelial Growth Factor (VEGF), Mucin 1 (MUC1) and Carcinoembryonic Antigen (CEA) characteristically associated with breast cancer are typically diagnosed with antibody-based immunochemistry or ELISA. However, the inherent averse of the antibodies for need-based modifications, besides their inherent limitation exhorts for exploration of alternate options. The authors used aptamers in an ingenious manner to detect the HER2 with detection linearity in the range of 0.1–100.0 ng/mL and a LOD of 0.08. ng/mL Further, in this study the authors also address the issue related to random entanglement and aggregation when not anchored properly and thus hindering their proper binding with the analyte. The authors anchored the aptamer on a Tetrahedral DNA nanostructure (TDN), formed by hybridization of four ss DNA sequences (aptamer being a part of the fourth sequence itself forming a pendent like structure) which themselves are thiolated at 3 ends. These thiolated ends cement the TDN-aptamer firmly on the gold electrode via thiolgold interaction. The recognition of HER2 is carried out in two steps, first the anchored aptamer recognizing the HER2 and capturing it on the electrode surface (as the whole apt-TDN, is anchored on electrode vis Au-S bond). In a separate step another aptamer recognizing a different epitope of HER2, and HRP are loaded on a Mn3O4/Pd@Pt nanocarrier to form nanoprobe 1. This nanoprobe 1 then binds with the already captured HER2 on electrode surface. To further have an accentuated signal amplification, a cDNA (complementary to sequence in nanopoprobe 1) is tagged with Pd@Pt/HRP, nanoprobe 2 and is added to the surface which joins the already formed complex with DNA hybridization with nanoprobe 1. This structure now has three nanozymes working in 17
tandem for signal generation and amplification viz. the Mn3O4 nanoflowers, Pd@Pt nanoflowers and HRP, by catalyzing the oxidation of hydroquinone (HQ) with H2O2 to benzoquinone (BQ) (Figure 7). The response in recorded by differential pulse voltammetry (DPV) for more sensitive detection. The test was able to perform in serum samples with a error within 10% when compared with standard ELISA, marking the potential of the biosensors for clinical diagnosis [54].
Figure 7: Principal of the electrochemical dual-aptamer biosensor for HER2 detection (HRP: horseradish peroxidase; GE: gold electrode; TDN: tetrahedral DNA nanostructure; MCH: 6mercapto-1-hexanol; HQ: hydroquinone; BQ: benzoquinone; DPV: differential pulse voltammetry). (Reprinted with permission from ref. 54. Copyright 2019, Royal society of chemistry).
18
Table 1: A A comprehensive list of nanozyme and aptamer based biosensors and their attributes. S.NO.
DETECTION MARKER
DETECTION TECHNIQUE
1.
Human norovirus (NoV) Zearalenone (ZEN) Lead (Pb2+) ions
colorimetry
Ochratoxin A (OTA) Ochratoxin A (OTA)
colorimetry
Pseudomonas aeruginosa
electrochemical
2. 3.
4. 5.
6.
colorimetry Surfaceenhanced Raman spectroscopy (SERS)
colorimetry
DETECTION RANGE 20-3,300 norovirus/mL
10-250 ng·mL−1 0.13–53.33 nmol/L
LIMIT OF DETECTION
REFERENCE
3 viruses in 100 µL 10 ng·mL−1
[44]
0.07 nmol/L
[46]
[45]
0.1–10 0.08 ng/mL ng/mL 1.25-250 nM 0.069 nM
[47]
60 CFU/mL
[50]
[27]
19
7.
kanamycin
electrochemical 0.1-60 nM
0.06 nM
[53]
8.
Mucin 1 (MUC1)
16 fg mL−1
[55]
9.
Human Epidermal Growth FactorReceptor 2 (HER2)
electrochemical 100 fg mL−1 to 1 ng mL−1 electrochemical 0.1–100.0 ng/mL
0.08ng/mL
[54]
Conclusions and future directions In summary, in the current review, we have given a brief overview of the intrinsic peroxidase-like activity of nanoparticles (nanozymes) and also through a light on application of nanozymes in combination with aptamers for biosensing applications. The aptamer-nanozyme system has wider utility and has shown its value to detect variety of analytes ranging from small molecules to whole cell. Further, adaptation of this system on to an electrochemical sensing platform has further increased the sensitivity and low-end detection limit. The aforementioned advances in this field have given a strong foundation to develop robust, rapid, sensitive and cost-effective point-of-care
20
device similar to lateral flow in near future that can be used as a Target Product Profile 1 (TPP-1) diagnostics[56]. Acknowledgments T.K.S. wishes to thank the Department of Biotechnology, Govt. of India for DBT-Innovative Young Biotechnologist Award (BT/010/IYBA/2016/10). T.K.S. also thanks Translational Health Science and Technology Institute (THSTI) for providing core grant funds to support.
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