Alessandra Vinelli, Manuele Onofri and Giampaolo Zuccheri
8.2 ENHANCEMENT METHODS BASED ON ENZYMATIC (OR CATALYZED) REACTIONS(OR CATALYZED) REACTIONS
A number of methods have been proposed in which an enzyme is finally co-immobilized with the target on the sensing spot. The enzyme, often a peroxidase or a phosphatase, will convert a substrate into a to-be-sensed product. The accumulation of this product as a result of a limited number of hybridization events is the signal enhancement strategy (see Scheme 8.1).
Other enzymes have been used as well. Glucose oxidase has been employed several times as the enhancer label in sandwich assays. Xie and coworkers obtain a 1 fmol/l detection limit (working with 1 µl samples) by using an electroactive polymer (containing osmium) as the charge carrier from glucose oxidase to the electrode (Xieet al.2004).
Target HRP-STV
Ty bio
Ty
Ty Ty
Ty
Ty
Ty Ty Ty Ty Ty Ty
Ty
Scheme 8.1 Scheme of the tyramide-peroxidase signal enhancement. Detection of the products (fluorescent tyramine or others, from different substrates of the enzyme) can be done with optical or electrochemical methods. The target DNA can be biotinylated through its amplification using biotinylated primers (as showed in the scheme) or using a secondary complementary oligonucleotide (sandwich approach).
Successive binding of peroxidase-streptavidin chimera (HRP-STV) and treatment with tyramide conjugated with a fluorophore (Ty) leads to very quick binding of the many fluorophores to the region of target binding.
Biochemical and nanotechnological strategies for signal enhancement 115
8.2.1 Peroxidase to enhance the signal of nucleic acids detection Tyramide-mediated fluorescence signal generation
As an example of the basic principle,tyramide signal amplification(TSA) is presented. This commercial method (commonly used to enhance the signal due to many different types of molecules, not only nucleic acids) binds a biotinylated target on the sensing spot. Peroxidase-conjugated streptavidin is then bound to the target. Fluorescein-conjugated tyramide is used as an enzyme substrate to produce an insoluble fluorescein derivative, the accumulation of which is then measured. Jin and coworkers applied tyramide signal amplification on DNA microarray for the multiple detection of six waterborne pathogen causing diarrhoea in human (Jinet al. 2008). Coupling multiplex PCR and TSA-Cy3 labelling method they could detect 103CFU/ml of each pathogen species, thus the use of tyramide signal amplification represents an effective alternative to the direct labelling with fluorescent dyes.
Peroxidase as an enzyme label in a sandwich assay
Exploiting the same strategy as in the previous example, Alfonta and coworkers directly bonded one peroxidase molecule to a secondary oligonucleotide (Alfonta et al. 2001). This is hybridized to the probe-immobilized target DNA on an electrode surface. In the presence of hydrogen peroxide added after rinsing of the unbound peroxidase, 4-chloronaphtol is converted into an insoluble compound by the peroxidases, thus leading to a measurable hindrance to the electron flow through the electrode.
In a similar assay, which exploits the same signal enhancement, Ostroff and coworkers detect the presence of the increasing amount of insoluble product through a change in reflectance of a surface (Ostroffet al.1999). The reported detection limit is 5 pmole/l (150 amol/sample).
Electrogenerated luminescence or electrogenerated precipitate formation as a signal enhancement induced by an electroactive intercalator
Doxorubicin is an intercalator molecule that binds at GC pairs along dsDNA. If a target DNA is immobilized on an electrode surface by a capture oligo in the presence of doxorubicin, then the doxorubicin molecules immobilized close to the electrode can undertake electrochemical reduction (Patolskyet al.2002). Reduced doxorubicin can be cycled by dissolved molecular oxygen which is, in turn, reduced to hydrogen peroxide.
According to this strategy, hydrogen peroxide accumulates over time as a result of DNA hybridization. Its presence is revealed by soluble peroxidase, which uses hydrogen peroxide (reducing it to water) to convert luminol to 3-aminophtalate which is chemiluminescent. Alternatively, in the presence of 4-chloronaphtol, an insoluble product is accumulated on the surface of the electrode. The accumulation of this product is detected by the hindrance to the charge transfer through the electrode.
The reported detection limit of this strategy is 10−11M. Even though this type of methods seems to perform well, using intercalators yields to the possibility that portions of double-stranded nucleic acids not related to the recognition event might bind the intercalator, either competing or leading to non-specific positive signals. Several different electroactive intercalators are available and have been used for similar (sometimes unenhanced) detection methods.
8.2.2 One to several instances of alkaline phosphatase for the electrochemical detection of nucleic acids
Detection by redox of an organic mediator
In a sandwich-type detection, the analyte RNA molecule can be immobilized on a sensor spot by an adsorbed oligonucleotide probe. Other regions of known base sequence of the target can be exploited to
bind one to several secondary biotinylated oligonucleotides (see Scheme 8.2). These can then bind streptavidin-conjugated alkaline phosphatase molecules. In the presence of alkaline phosphatase, p-aminophenyl phosphate is converted to p-aminophenol, which thus accumulates over time in the case (and in the location) of RNA recognition and binding. p-Aminophenol can be oxidized to the corresponding quinoimide compound. The electric current derived from redox-cycling of this compound can be measured (implementing the chemoelectronic sensor as a series of interdigitated electrodes). The presence of the bound enzyme will make the redox current grow with time due to the progressive accumulation of the electroactive molecule, thus leading to signal enhancement. The method is straightforwardly implemented on a surface of a microfabricated device. Several application of alkaline phosphatase for enhancement of the signal due to 16S rRNA detection have been proposed (Elsholz et al.2006; Elsholzet al.2009; Walteret al.2011). Several pathogens, some of which might be found in drinking water and are possible bio-threat agents (E. coli,P. aeruginosa,E. faecalis,S. aureus,Y. pestis, B. anthracis andS. epidermidis) were identified in parallel using nanometric interdigitated gold array electrodes (Elsholz et al. 2006; Elsholz et al. 2009). Working at constant current and varying the potential of the interdigitated electrodes, the authors monitored the time necessary to chemical conversion of the substrate oxidized by the enzyme, which is directly proportional to the analyte concentration. A limit of detection 0.5 ng/µl was achieved forE. coliRNA.
The same type of signal enhancement (redox cycling of alkaline phosphatase products) has been proficiently employed for the detection of DNA too, using a quite similar electrochemical set-up and microfrabricated electrodes (Schienleet al.2004).
Detection by redox of a metallic mediator
In another use of alkaline phosphatase (AP) as a signal enhancing mediator, the hybridization event and the subsequent formation of a sandwich compound with an AP-labelled oligonucleotide is measured after the reduction of soluble Ag(I) ions to insoluble silver (Hwanget al.2005). Such reduction is undertaken by aminophenol produced by the reaction of AP on p-aminophenylphosphate (vide supra). The presence of even a few copies of the enzyme can lead to the reduction of a considerable amount of silver. Deposited silver is then detected via anodic electrochemical stripping.
Scheme 8.2 Schematic representation of the binding and labelling of a ss-RNA or DNA (long black strand) by a surface-tethered oligonucleotide, with subsequent (singular or multiple) labelling by oligonucleotides bound to alkaline-phosphatase (represented as a sphere).
Biochemical and nanotechnological strategies for signal enhancement 117
Due to the formation of a solid silver deposit, other techniques can be alternatively used to detect the hybridization event, such as a quartz crystal microbalance or a surface plasmon resonance detector, as both techniques are sensitive to the added mass on the surface. It can be foreseen that also optical absorbance properties (or light scattering) will change as well as the reduction proceeds. A detection limit of 10 zmoles (100 aM concentration) is claimed by the authors, as well as the specificity to detect single base mismatches (Hwanget al.2005).
More than one alkaline phosphatase per binding event: macroenzymatic labels
Munge and coworkers prepared carbon nanotubes coated with several protein layers containing alkaline phosphatase molecules together with streptavidin (Mungeet al.2005). Such nanotubes can be associated with oligonucleotides to be used in sandwich recognition assays. A functional nanotube is immobilized on the target which has been immobilized by the main capture probe (on a surface of an electrode or of a bead).
The authors claim that such high number of alkaline phosphatase molecules that are recruited by very few DNA-binding events will lead to the detection of as few as 80 copies of a nucleic acid (a concentration of 5 aM in the reported conditions).
8.2.3 Terminal transferase to grow DNA at the recognition site
Terminal deoxynucleotidil transferase (TdT) is a mammalian enzyme that extends the 3′-OH end of a single-stranded DNA in a template-free fashion. The incorporation of dNTP is relatively random, even though preferences can be found in the presence of different cationic cofactors. Homopolymers can be obtained by feeding the enzyme with a single type of nucleotides. It has been shown that TdT can be used to extend surface bound oligonucleotides (Chowet al.2005). The rationale for its innovative use as a possible enhancement technique resides from the consideration that a 3′ bound DNA probe oligonucleotide cannot be extended by TdT, while the target DNA that could bind to it should present a free 3′-OH amenable of TdT mediated extension. Extension would result in the accumulation of nucleic acid in a surface-bound state, and thus to the enhancement of a detection signal if this is proportional to the amount of DNA bound at a specific location on a surface (see Figure 8.1).
At our laboratory, experiments in solution analyzed by gel-electrophoresis showed that the reaction was efficient and relatively fast as to be a good candidate strategy. Higher molecular weight products were obtained starting from short 3′-OH free oligonucleotides. No extension was observed when the oligonucleotide had no free 3′-OH, showing that the specificity of the enhancement reaction could be as high as the specificity of target recognition by the probe, as we desired. Once attempted in a surface-bound format, the reaction resulted as less efficient leading to an enhancement factor of not more than 5, when the amount of DNA was determined by fluorescence measurements (via the non-specific binding of SybrGold to the surface-bound DNA). Alternative labelling strategies or detection techniques might prove more efficient.
8.2.4 Signal enhancement of fluorescence through the use of a nickase When a fluorescent probe is used for detecting a target (e.g. through a molecular beacon approach), the cleavage of the probe in case of hybridization can release the target in solution for subsequent binding events. In an implementation of this strategy using a nickase, Zheleznaya and coworkers used molecular beacons and a site specific nickase to obtain an enhancement factor of 100 (Zheleznayaet al.2006).
Very interestingly, researchers also noted some interference in the assay in case of the presence of extraneous DNA, that leads to a decrease in the signal (rather than to an expected increase due to non specific binding) probably due to the binding of the nickase to the extraneous DNA.
In a recent application, a nicking enzyme sensing assay was coupled with CdSe/ZnS quantum dots amplification for cymbidium mosaic virus detection (Chenet al.2010). A thiolated hairpin DNA probe labelled with biotin was immobilized on gold electrode via S-Au bond. The double strand loop of the hairpin contained the restriction site for the endonuclease BfuCI, the nicking enzyme. In absence of the target, the hairpin bounded on the surface was closed and the restriction enzyme could digest the loop. As a consequence, the oligonucleotide end labelled with biotin was released in solution and the avidin-QD conjugate could not bind. On the contrary, the presence of a target molecule in solution opened the hairpin, blocking the enzymatic digestion and leading to the binding of avidin-quantum dots conjugate to the biotinylated probe. The excess of QD was removed and the electrochemical detection was performed after a treatment with acid solution to dissolve quantum dots. Stripping voltammetric measurements of the Cd2+
ions were performed using anin situplated mercury film on a glassy carbon electrode. Using this indirect electrochemical measurements, the author reported the detection of 3.3×10−14target molecules.
8.2.5 RNase H as a target recycling operator for RNA-based sensors
Goodrich and coworkers implemented a method to recycle the (few) target DNA molecules by preparing a sensing surface with RNA oligonucleotide probes (Goodrichet al.2004). After binding with the target (and thus forming a RNA:DNA hybrid) such double strand can be the substrate for RNase H in solution. Its action
(a) (b)
T TdT dNTPs
Figure 8.1 a) Scheme of the use of terminal deoxynucleotidyl tranferase (TdT) for signal enhancement in DNA biosensors. TdT catalyzes the addition of nucleotides at the free 3′terminus of DNA. A target DNA (T) paired at a 3′-blocked, surface-bound probe can then be extended leading to the accumulation of more DNA on the recognition spot. b) background (lower trace, circle markers), target (middle trace, square markers) and enhanced (upper trace, triangle markers) fluorescence emission spectra for the TdT reaction signal enhancement on a biosensor surface. TdT enhancement was performed after target-DNA exposure onto a probe-functionalized gold-electrode. Later, the surface-bound DNA was removed from the surface by mercaptoethanol treatment (Demerset al. 2000) and the resulting specimens (no target DNA, target, target+TdT enhancement) were stained with SybrGold dye. The resulting fluorescence spectra (with intensity dependent on the amount of surface-bound DNA) measured an approximately 3-fold signal gain.
More specific detection methods could lead to an even larger enhancement factor.
Biochemical and nanotechnological strategies for signal enhancement 119
leads to the hydrolysis of the bound RNA oligonucleotide and to freeing the target in solution again, so that it can bind to another surface-immobilized oligonucleotide. Over time, all the specific RNA oligonucleotide probes are digested by RNase, thus leading to signal enhancement (for instance if read through surface Plasmon resonance or other techniques). The reported detection limit is 10 f M in a 13 µl sample volume.
An intrinsic weakness of this method, in our view, is that RNA is a fragile molecule, that can undergo hydrolysis due to a number of agents tha can be present in a complex analyte matrix. Such non specific hydrolysis could probably be detected and distinguished from the specific one, but it will nonetheless reduce the effectiveness and solidity of the proposed method.
8.2.6 Nucleic acid sequence-based amplification (NASBA)
The NASBA, also known as Self Sustained Sequence Replication (3SR) (Guatelli et al. 1990) is an enzymatic technique that amplifies an RNA molecule through a complex, though isothermal series of enzymatic reactions. Such technique is often used (also in commercial assay kits) for the pre-detection amplification of RNA target analytes. As depicted in Scheme 8.3 below, the different steps lead to the conversion of one RNA target into a DNA template for various rounds of transcription, leading to amplification. Briefly, the analyte RNA is paired to an oligonucleotide primer (P1) which is elongated to complementary DNA by reverse transcriptase. The RNA analyte paired to such DNA is hydrolyzed with RNaseH, so that an oligonucleotide primer (P2) can bind to the just-synthesized DNA, providing a substrate for reverse transcriptase. Reverse transcriptase produces a full dsDNA molecule. As P1 is made to include a T7-RNA polymerase promoter site, the produced dsDNA can serve as template for the synthesis of many copies of RNA complementary (antisense) to the target RNA sequence by T7-RNA polymerase present in the same mix: this is the true amplification step. In the mix, P2 can now bind to the produced antisense RNA, start the same process again using P2 and reverse transcriptase to start with using the antisense RNA and ending with producing many copies of sense RNA to feed the cycle.
P1
T7 promoter
RNA target
P2
P2
P1 antisense RNA
Rev. Transcr. Rev. Transcr. T7 RNA pol.
Rev. Transcr.
Rev. Transcr.
T7 RNA pol.
RNaseH
RNaseH
Scheme 8.3 Depiction of the NASBA procedure for nucleic acids amplification. RNA strands are represented as dashed lines, while DNA is solid. Black lines are the‘sense’sequences while gray lines are the‘antisense.’ The dot in primer P1 only shows the separation between the target sequence and the added T7 promoter. The NASBA procedure works in a single solution kept in isothermal conditions where all the depicted reactions work in sequence with the result of producing large amounts of antisense RNA in case the sense RNA is present.
In certain implementations, the NASBA has been proficiently employed towards the signal enhancement through its surface implementation (Edman et al.2006). The very high amplification factor of solution implementations of this reaction (detection of 50 copies of nucleic acid) will probably lead to interesting surface-bound applications. In recent applications, NASBA has been successfully applied on microarray systems. Scheler and coworkers optimized NASBA protocol in order to obtain a chemical-modified RNA product (Scheler et al.2009). On it, post-amplification labeling with fluorescent dye was carried out and detection was performed with microarray technology. A lateral-flow based platform for B. anthracis amplicon detection was developed by Carteret al. (Carteret al.2007). In this application target RNA is not directly labeled but is hybridized in a sandwich formed by an immobilized DNA capture probe and a microsphere-conjugated detection probe. Accumulation of the microsphere in correspondence of the capture feature produce a colorimetric signal which is visible at naked-eye and easily detected at low concentrations using widely available flatbed scanners. The reported sensitivity for the system is sub-femtomolar.
Transcription Mediated Amplification (TMA) is a very similar (and commercially available) amplification procedure that exploits essentially the same mechanism but making use of a different type of reverse-transcriptase and thus avoids the use of RNaseH (Hill, 2001; Chelliserrykattil et al. 2009;
Bachmannet al.2010; Raoet al.2010).
Even though direct surface implementations of NASBA to lead to post-hybridization signal enhacement are still somewhat lacking, the technique bears some interest for the scope of this review as the reaction can be performed directly in the hybridization/sensing chamber of the biosensor and thus practically work in an integrated fashion with the biosensor, without the need of introducing alternative or additional instrumentation or procedures, other than the addition of the reaction mixture and incubation (usually for 1 hour).
8.2.7 Strand displacement amplification
Along the same line of reasoning of NASBA, strand displacement amplification (SDA) is another strategy towards nucleic acids amplification that can be quite effortlessly integrated in the biosensor detection compartment. It is an isothermal method to amplify DNA that has been proposed in 1992 by Walker and coworkers (Walker et al. 1992; Walker et al. 1992). It is a rather complex mechanism based on consecutive events of restriction and amplification. The use of four primers is requested (two amplification primers and two restriction primers). Nowadays, kits for TMA are commercially available.
Even though the reaction seems to perform well, it quickly amplifies nucleic acids and it could possibly be integrated in biosensors-based detection, all the conditions and rules for the design of the 4 primers and the avoidance of amplification artifacts do not seem as mature as for other techniques (as seen from some attempts performed during the DINAMICS EU research project).
SDA has been used for pathogen detection in solution (Bachmannet al.2010). Recently SDA has been coupled with piezoelectric detection for the real time monitoring of human cytomegalovirus (Chenet al.
2009). Quartz crystals have been modified with a specific DNA capture probe: during the strand displacement amplification, products accumulate on the crystal surface. The limit of detection presented is 1 ng/ml.
8.2.8 Loop mediated isothermal amplification (LAMP)
LAMP is a method proposed by Notomi and colleagues (Notomiet al. 2000). The LAMP mechanism, like SDA, is based on the strand displacement activity of DNA polymerase. For the reaction, a set of four specific primer is necessary. Primer design is a key point of this complex mechanism: the four primers hybridize to six different specific regions on the template DNA, separated by a defined distance on the Biochemical and nanotechnological strategies for signal enhancement 121
sequence. The external primers duplicate the dsDNA and displace the products of the internal primers, which bear loops. The looped product, together with the loop primers allow the progress of multiple polymerizations
sequence. The external primers duplicate the dsDNA and displace the products of the internal primers, which bear loops. The looped product, together with the loop primers allow the progress of multiple polymerizations