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. 2024 Dec 25;10(3):1788–1796. doi: 10.1021/acssensors.4c02492

Pathogen-Specific Electrochemical Real-Time LAMP Detection Using Universal Solid-Phase Probes on Carbon Electrodes

Martin Trotter †,*, Andreas Schreiber †,‡, Dominic Kleinknecht †, Zahra Bagherian †,‡, Felix von Stetten †,‡, Nadine Borst †,‡,*
PMCID: PMC11960684  PMID: 39721602

Abstract

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Epidemic infections and spreading antibiotic resistance require diagnostic tests that can be rapidly adopted. To reduce the usually time-consuming adaptation of molecular diagnostic tests to changing targets, we propose the novel approach of a repurposable sensing electrode functionalization with a universal, target-independent oligonucleotide probe. In the liquid phase covering the electrode, the target sequence is amplified by MD LAMP (mediator-displacement loop-mediated isothermal amplification) releasing a generic methylene blue-labeled mediator, which specifically hybridizes to the solid-phase probe. To demonstrate the universality of the approach, two different pathogens, Staphylococcus aureus (crude lysate) and Treponema pallidum, are detected using the same solid-phase probe. The reactions reach a limit of detection of 1 × 103 and 4 × 102 copies per reaction within 30 min, respectively. The solid-phase probes carry a carboxymethyl aniline modification to form covalent C–C bonds on low-cost carbon electrodes. Maximum surface coverage and maximum hybridization signals are observed at grafting concentrations of ≥2 μM solid-phase probes. Successful detection of spiked target DNA in real swab samples and with three different commercial amplification buffers proved the broad applicability of this assay approach. The electrochemical MD LAMP is fast, compatible with dsDNA targets, and requires only minimal adaptation of an established amplification method. It is easily transferable to existing analytical electrochemical platforms, allowing the consumable to be synergistically used for different targets. The suggested approach of repurposable functionalized electrodes can also be considered to increase the preparedness for future epidemic or pandemic outbreaks as well as rapidly evolving resistance patterns or variants.

Keywords: real-time LAMP, DNA testing, universal electrochemical detection, solid-phase probes, carbon electrode, point-of-care, signal-on, MRSA

Point-of-care (POC) molecular testing is a growing market and of particular interest for the detection of infectious diseases that require immediate action such as targeted treatment and/or measures against spreading. An example is the detection of methicillin resistant Staphylococcus aureus (MRSA) that may lead to isolation of the patient. Further benefits are expected for medical care in remote settings, where fast access to well-equipped laboratories is complicated.1 Highly desired molecular POC testing features sensitive and specific real-time signal-on detection. In addition, the test should be fast (<30 min), based on minimal robust and miniaturized instrumentation, and allow for multiplex detection. New emerging diseases or development of new resistances require the ability to easily adapt the test to new markers.2 The necessity for such developments gives rise to the design of novel assay concepts and sensing technologies, including optical and electrochemical detection methods.3 Optical detection is a well-established technique, commonly employed in the field of point-of-care (POC) testing.4 However, electrochemical detection has shown great potential for use in molecular testing5 especially when miniaturization and robustness are of relevance.6,3 Consequently, two of the four companies (namely Aptitude Medical and Cue Health) that received an FDA emergency use authorization for their at-home molecular tests employ electrochemical readout.7

Isothermal amplification methods have gained attention as a compelling substitute for classical PCR and are particularly relevant for miniaturized POC systems, because they entail reduced instrumental complexity. Especially, the loop-mediated isothermal amplification (LAMP)8 is notable for the abundance of publications it has garnered, primarily owing to its amplification speed and sensitivity.9 The most common electrochemical LAMP detection approach is certainly the homogeneous, label-free detection of the amplification products that relies on the intercalation of redox molecules in the generated double-strand, which causes a decay in signal (signal-off behavior).10−12 Even though these methods are easy to adapt, as they do not require probe-design or the functionalization of electrodes, it is difficult to discriminate between specific and unspecific amplification or between different targets. This makes the method prone to false-positives and not suitable for multiplex detection, even though melting curve analysis can increase specificity.13,14

For multiplex detection, probe-based assays are necessary. Unlabeled solid-phase primers or probes are used to either enrich redox molecules, e.g., by intercalation into the double-strand15 or by incorporation of labeled nucleotides into the amplicons,16 or to electrostatically repel redox molecules17 upon target amplification and binding. Labeled solid-phase probes change their electron transfer characteristics upon hybridization of amplification products.18 However, these approaches acquire the signal after the amplification in end-point detection and partially require washing and buffer exchange steps.

Lately, probe-based electrochemical real-time LAMP detection was demonstrated by prehybridizing a redox-labeled probe to the capture probe that is displaced during amplification of the target sequence, leading to a decaying signal for positive reactions.19

These kinds of probe-based assays are quite specific and sensitive but require the lengthy adoption of electrode functionalization for each new target sequence. This increases the time and effort for development and approval, which is especially critical for newly emerging diseases. Attempts to generate universal electrode functionalization have been made (see Table S1) but have been limited to single-stranded targets, high limits of detection (LoD), or long reaction times.20−27

A recent approach, called Iso-E-Codelock,28 addresses some of these shortcomings by enabling real-time electrochemical signal-on monitoring of LAMP on universally functionalized electrodes. It utilizes a 3- or 4-way junction probe that contains a target-specific portion and releases a labeled generic probe, which hybridizes to universal solid-phase probes. The approach provides sensitive results within 120 min of reaction time.

Here, we present an electrochemical real-time assay that combines the desired outlined characteristics by combining signal-on behavior, with the speed, specificity, and sensitivity of probe-based LAMP. The presented electrochemical mediator displacement LAMP (eMD LAMP), first submitted back to back with ref (28), allows fast reaction times (<30 min) with only minimal adaptations to the standard LAMP reaction. Unlike the Iso-E-Codelock system,28 which requires the introduction of three oligonucleotides to form the probe, the eMD LAMP approach relies on only a slightly extended Loop primer by a sequence that binds the methylene blue (MB)-labeled generic mediator oligonucleotide (Scheme 1). The target-specific participation in the amplification of the Loop primer results in the displacement of the mediator,29 which hybridizes to the solid-phase probe, called the universal reporter (UR). The MB label, which was chosen for its good stability30 and low interference13 with the reaction, accumulates at the surface and leads to an increasing detection signal.

Scheme 1. Reaction Scheme of the Herein Described eMD LAMP.

Scheme 1

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(A) DNA amplification via LAMP leads to characteristic dumbbell-like amplification products. The mediator probe that consists of a target-specific primer sequence supplemented by a generic sequence (Medc), to which the labeled generic mediator is hybridized, anneals to the amplification product. (B) Upon extension, the mediator probe becomes involved in further amplification reactions. (C) During these reactions, the extension of complementary primers leads to the displacement of the labeled mediator. Hybridization of the mediator at the universal reporter enriches the label at the electrode surface, leading to a signal increase for positive reactions. The scheme starts with already formed dumbbell-like amplification products (for prior reaction steps see Supporting Information). The further steps show the most simple of several possible reaction pathways. Primer or probe sequence portions are colored and labeled, where "c" stands for "complementary”. Arrowheads indicate at which end the primers are extended.

Beneficial for the production and functionalization of electrodes in large standardized batches, our approach allows for the one-time optimization of the solid-phase detection reaction. Oligonucleotides for target identification could be part of the liquid-phase reagents. The hardware components of a test (functionalized electrode array, cartridge) can thus be universal, making it easier to repurpose the components to the actual need or to synergistically serve different needs.31 This is expected to reduce the effort, time, and cost of development and approval and increase preparedness for future pandemics.2

In this work, URs are immobilized on screen-printed carbon electrodes. In terms of production costs and sustainability, the approach is advantageous compared to the commonly used but expensive gold electrodes. We optimized the UR electrode coverage, UR design, and mediator concentration to increase the signal-to-noise ratio (SNR) and determined the analytical performance. The universality of the approach is demonstrated by the detection of two different DNA targets with the same reporter sequence: the mecA gene of S. aureus, which indicates resistance to methicillin, and Treponema pallidum, a bacterium that causes the neglected tropical disease yaws.

Experimental Section

Materials

Primers and probes for the detection of the mecA gene in methicillin-resistant S. aureus were adopted from Nanayakkara and White.32 An additional self-designed LoopB primer with and without a supplemented mediator binding site (Medc sequence) was introduced. Crude lysed MRSA cultures in 20 mM Tris – HCl (pH 7.6) were used for mecA detection (for details see Supporting Information).

T. pallidum was detected using a LAMP assay for the polymerase I gene (polA)33 using primers and a mediator that were optimized for optical MD LAMP.34 As in the aforementioned publications, circular plasmids of 2.75 kbp total length (Eurofins, Germany) containing 300 bp clones of the target sequence were added to positive control reactions (PTC).

All oligonucleotides were purchased from Biomers (Ulm, Germany) in HPLC grade (sequences listed in Tables S2–S4). The other reagents and their sources are listed in the Supporting Information.

Instrumentation

Electrochemical experiments were mainly conducted on a Leo prototype on 48 well plates (see Figure S1) that contain screen-printed carbon working and counter electrodes as well as silver pseudoreference electrodes in each well (Easy Life Science/Quattrocento, France). The prototype allows the analysis of all 48 wells in parallel using square-wave voltammetry (SWV), while controlling the temperature.

If not stated otherwise, SWV was acquired each 20 s from −50 to −550 mV vs the pseudo Ag reference with f = 150 Hz, 25 mV square-wave amplitude (i.e., 50 mV peak-to-peak), and 1 mV step-width.

SWV data was evaluated with a custom Python script that determined the peak current (ΔiP). Data aside from the peak was used to fit a third-order polynomial as a baseline that was subtracted from the original curve, which was then analyzed for its maximum current and corresponding potential (see also Figure S2 for details).

A Gamry 1010 B potentiostat was used for cyclic voltammetry (CV) and chronocoulometric measurements. Data evaluation was conducted using the Gamry Echem Analyst.

Electrode Functionalization

Universal solid-phase probes were immobilized to the carbon working electrode via electrochemical grafting of oligonucleotides that featured a 4-carboxymethylaniline (CMA) modification at their 3′- or 5′-end following a protocol of Corgier et al.35 The oligonucleotides are commercially available with CMA-modification and are ready to use as follows:

The 48 well plates were initially washed by immersion in 2-propanol for 5 min and 3× in DI-water for 5, 3, and 5 min. The immobilization solution was prepared on ice to contain final concentrations of 15 mM NaNO2, 15 mM HCl, and typically 2 μM CMA-modified solid-phase probes. The solution was allowed to react on ice for 20 min to start the diazotation process. Afterward, 40 μL of the solution was loaded into the wells of the 48 well plate on ice. For electrochemical grafting, three consecutive sweeps of SWV were conducted in the Leo prototype between 0.5 V and −1 V. The SWV was essentially a linear staircase voltammetry with a square-wave amplitude of 0 V and a frequency of 50 Hz (corresponding to 75 mV/s).

The wells were subsequently washed with 50 μL of 1 × PBS for 3 min and then blocked with 50 μL of 1% BSA, 5× SSC and 0.1% SDS for 60 min at 42 °C. The final washing steps consisted of 2 times with 1× PBS and finally DNase-free water for 5 min, each. The well plate was dried in a desiccator and stored dry until use.

Characterization of Electrode Functionalization

The optimum concentration of CMA-modified oligonucleotides was investigated by determining the number of immobilized DNA molecules and the number of hybridized MB-labeled mediators.

Therefore, the concentration of CMA oligonucleotide in the immobilization mix was varied between 0.5, 1, 2, 5, and 10 μM. Deviating from the protocol above, the electrodes were not blocked but just washed 2× with PBS and directly used for characterization. The number of immobilized oligonucleotides was determined according to the protocol of Steel et al.36 In short, chronocoulometry was conducted in Tris-buffer (pH 7.4), first in the absence and then in the presence of 50 μM Ru(NH3)6Cl3 (RuHex), by stepping from +0.15 V to −0.45 V. Accompanying CV measurements allowed an estimation of the WE surface area from the peak of the diffusive RuHex molecules.

After washing again with PBS, 40 μL of hybridization solution consisting of 0.2 μM mediators in 1 × isothermal amplification buffer II, 1 mM MgSO4, and 5% glycerol was loaded per well. Hybridization at room-temperature was first monitored by conducting SWV with the Leo prototype and afterward analyzed with the Gamry potentiostat performing CV measurements in surface mode to account for the surface confinement of the MB labels and SWV measurements. From the CV signals, the number of hybridized MB-labeled mediators was calculated according to Faraday’s law p = Q/nF, where Q is the integrated MB reduction peak, F is the Faraday constant, and n is the number of transferred electrons (n = 2 for methylene blue).

Hybridization and LAMP Reaction Buffers

Hybridization reactions and LAMP reactions were always conducted in volumes of 40 μL. Typically, the reaction mix consisted of 1 × Isothermal Amplification Buffer II, 320 U/mL Bst 3.0 polymerase, 4 mM MgSO4, 1.4 mM dNTPs, 0.2 μM F3 and B3 primers, 1.6 μM FIP and BIP primers, and 0.8 μM Loop primers supplemented with DNase-free water. The sum of unmodified Loop primers and LoopMedc primers was 0.8 μM, and the ratio of LoopMedc primers to mediators was 2:1 in any case. Typical concentrations were 0.4 μM unmodified Loop primer, 0.4 μM LoopMedc primer, and 0.2 μM mediator.

Mediator Displacement Reaction for Optimization

Mediator displacement reactions (Scheme S2) were conducted for initial parameter optimization, focusing on the displacement reaction of the mediator by primer extension and excluding the influence of the target amplification reaction.

The reaction mix contained polymerase, dNTPs and MgSO4, but no target DNA and only the oligonucleotides directly involved in the mediator displacement reaction, i.e., 0.4 μM of a general LoopMedc primer featuring binding sites for the mediator and an additional primer, and 0.2 μM mediators. After a sufficient initial incubation time at 65 °C to establish equilibrium, 0.6 μM displacement primers were added to displace the mediator upon extension by polymerase. This should simulate a positive reaction (quasi-PTC), while wells in which no additional primer was added represented quasi-NTC reactions. As a reference for the maximum achievable signal, wells were loaded with reaction mix that contained free mediators only. The reactions before and after primer addition were monitored in a Leo device. For primer addition, the plates were removed from the Leo and placed on ice to allow for comparable displacement reaction conditions.

Real-Time LAMP Reaction and Analysis

All real-time LAMP reactions were conducted at 65 °C. For the determination of the LoD, a dilution series of crude lysate from MRSA or target DNA for T. pallidum DNA was analyzed in 8 or 3 technical replicates, respectively. The LoD was calculated by Probit analysis using IBM SPSS Statistics 29 (IBM, Chicago, USA). The time to positive (ttp) of a reaction was determined by forming the derivative of the filtered signal (cubic Savitzky-Golay filter of window size 7) and determining its maximum using a custom-made Python script (more info in the Supporting Information).

To demonstrate that the eMD LAMP works also for other common LAMP reaction mixes, T. pallidum was not only amplified in the above-mentioned mix containing Bst 3.0 but also with Bst 2.0 warm-start polymerase and for different buffers: 320 U/mL Bst 2.0 was combined with New England Biolab’s Isothermal Amplification Buffer and 5 mM MgSO4, while the other components and their concentrations remained unaffected. Alternatively, 320 U/mL Bst 2.0 warm-start polymerase was combined with Mast Diagnostica’s RM MPM Buffer that already contained dNTPs and that was not supplemented with additional MgSO4.

The influence of real nasal swabs on the amplification and detection reactions was investigated by employing samples from three volunteers. After self-swabbing their nostrils by wiping the nasal atriums for 5 s each using a FLOQSwab (Copan, Italy), the swab was inserted in a 1.5 mL Eppendorf tube filled with 250 μL of MSwab transport medium (Copan, Italy) and agitated for 30 s. Each eluate was aliquoted in two tubes. 48.7 μL of eluate was then either spiked with 1.3 μL of lysed MRSA cells (2.1 × 104 cells per tube) solved in 20 mM Tris buffer (PTC) or with 20 mM Tris buffer (NTC). As controls, PTC and NTC reactions were also prepared in MSwab medium and in RNase/DNase-free water without a real nasal swab sample. All tubes were subjected to heat lysis for 3 min at 95 °C using a BioShake iQ (BioShake, Germany). Afterward, the samples were analyzed in triplicates. Each 40 μL reaction mix contained 8 μL sample, i.e., 3.3 × 103 spiked MRSA cells per reaction.

Results and Discussion

Optimization of UR Concentration for Electrode Functionalization

Electrode functionalization must be stable at temperatures of 70 °C over an ideally wide potential range. Furthermore, the solid-phase probe layer should allow high hybridization efficiency and electron transfer between the label and electrode. Frequently employed grafting of aryl diazonium salts with subsequent coupling of aminated DNA via carbodiimide cross-linking results in DNA layers that are very stable at high temperatures and under various reaction conditions owing to the formed C–C bonds.37 However, this two-step grafting process is prone to forming multilayers, affecting electron transfer.37 For assays that rely on the enrichment of a labeled probe at the electrode as intended in our case (Scheme 1), the grafting of a monolayer of solid-phase probes is critical to ensure a defined distance between the label and electrode for a reproducible electron transfer.

Therefore, we decided to follow a one-step electrografting protocol using solid-phase probes previously coupled to the diazonium precursor CMA.35 Besides the significantly shortened time for surface functionalization, we expect that the coupled oligonucleotide sterically hinders the formation of multilayers during grafting.

To determine the optimum surface functionalization for a maximum hybridization signal, different concentrations of the CMA-modified UR were electrografted. Quantification of immobilized URs by RuHex measurements confirmed that the surface coverage increases with an increasing concentration of CMA oligonucleotides for the lower concentrations. At concentrations of 2 μM and above, the surface coverage reached saturation at about 5 × 1010 to 5.8 × 1010 molecules (Figure 1). Referred to an electrode area of 3.6 ± 0.1 mm2 that was estimated from the CV peak of diffusing RuHex, this corresponds to a density of 2.4 ± 0.1 pmol/cm2 for 2 μM CMA oligonucleotides. The values are corrected by subtraction of the signals acquired for nonfunctionalized electrodes ((2.5 ± 0.2) × 1010 molecules). The correction has little practical consequence. In any case, the density is rather low and should allow the realization of an electrochemical DNA sensor with high hybridization efficiency close to 100%.38,39 Additionally, the observation of the saturation behavior indicates the formation of a monolayer of URs and the determined density suggests a submonolayer, as a monolayer of diazonium molecules has been determined to be at about 250 pmol/cm2.40 It is likely that the already coupled oligonucleotides sterically hinder multilayer formation, as described for other blocking groups at the para-position of diazonium.41,42

Figure 1.

Figure 1

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Surface coverage of the working electrode becomes maximal for immobilization mixes that contain 2 μM or more CMA oligonucleotides. Hybridization of MB-labeled mediators confirm accessibility of the probes as their number scales in dependence on immobilized URs. n = 2, asterisk n = 1.

Indeed, the following hybridization of 0.2 μM MB-labeled mediators at 30 °C showed a comparable signal dependency, indicating that the immobilized URs were accessible. The quantification of hybridized mediators indicated saturation between 4 × 1010 and 5 × 1010 molecules. For nonfunctionalized electrodes, no detectable peaks were identified, indicating that unspecific adsorption of MB-labeled mediators is negligible.

As a consequence, 2 μM CMA oligonucleotides, the lowest concentration at which the signal reached saturation, were chosen as the concentration for further electrode functionalization.

Oligo Design Optimization for Maximal Hybridization Signals

Further investigation of the hybridization reaction revealed that the signal decreased with increasing temperature (data not shown). Possible reasons could be melting of the mediator from the UR and the temperature dependency of SWV signals. Both factors were considered for signal improvements, starting with the latter:

In good approximation, the frequency corrected SWV signals (ΔiP/f) become maximum, when the SWV frequency equals the electron transfer rate of the redox label,43 which has practical implications for rational sensor design.44 As the transfer rate increases with the temperature, the optimum frequency also increases. A signal that decreases with increasing temperature thus indicates that the SWV frequency was chosen to be too low. However, the Leo prototype used allowed a maximum frequency of 150 Hz. As a consequence, a 5T-spacer was introduced between CMA modification and the UR sequence to reduce the electron transfer rate and better match the limitations of the potentiostat.

As hypothesized, mediator displacement reactions at 65 °C resulted in higher SWV signals with UR5T that contained the spacer in comparison to the UR0T without a spacer (Figure 2). Signal acquisition during cooling from 65 to 30 °C resulted in decreasing signals for UR5T but increasing signals for UR0T. The result underlines that the observed behavior during heating or cooling is mainly caused by the temperature dependency of the analysis method, rather than by oligonucleotide melting. This emphasizes that determining the melting temperatures (Tm) from SWV data is complicated and may require control reactions for correction.

Figure 2.

Figure 2

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Introducing a 5T-spacer sequence to the UR improved the SWV signal at 65 °C for the given SWV frequency restriction (150 Hz). The figure shows the SWV peak currents over time for a mediator displacement reaction, where the mediator displacing primer was added between 33 and 38 min. Cooling the reaction chambers from 50 min onward illustrated the difference in temperature dependency for UR with and without spacer. Data points represent the mean and standard deviation of n = 3.

Investigating the Tm (respectively binding energy) is initially necessary for optimal UR design since it affects the SNR. The design rules for optical MD LAMP specified that the binding energy of the mediator needed to be higher at the LoopMedc primer than at the UR (see Scheme 1) in order to reduce the baseline signal, which resulted in a shorter hybridization site for the mediator at the UR.29 However, this is true if all oligonucleotides are in solution, whereas in our case, the UR is bound to the surface, which is expected to lower the Tm.45 We thus used LoopMedc and UR with mediator hybridization sites that were fully complementary. In order to increase the binding enthalpy, we introduced three LNA modifications in the mediator binding sites of LoopMedc and/or UR and investigated the resulting four different combinations. The best SNR was achieved for the combination of UR and LoopMedc where neither was modified with LNA (for details, see Figure S3).

In order to further assess the reproducibility of surface functionalization and hybridization with the optimized UR, six electrodes were grafted with 2 μM UR5T followed by a hybridization reaction with 0.2 μM mediators at 65 °C. The hybridization signal after 30 min was (2.4 ± 0.2) μA. The observed relative standard deviation of only 8% demonstrates good reproducibility of the grafting and hybridization process.

Influence of MB Label Position and Mediator Concentration

Previous publications reported that the modification of primers can affect amplification efficiency.29,32,46 For this reason, Becherer et al. only replaced a small proportion of the Loop primers with LoopMedc primers and mediators. Furthermore, as MB is able to intercalate in DNA, it may play a role, whether the label is positioned at the 3′- or the 5′-end of the mediator.

We therefore analyzed real-time LAMP reactions (target: 104 copies of the mecA gene) that contained different concentrations of 3′MB-labeled and 5′MB-labeled mediators, respectively. The ratio of mediator to LoopBMedc primer remained unchanged and was 1:2. The sum of LoopBMedc and LoopB was always 800 nM.

Reactions with 3′MB-mediator and 5′MB-mediator were always conducted in wells that contained 5′CMA-UR5T and 3′CMA-UR5T, respectively, so that the distance of the label to the electrode surface remained comparable.

Reactions with 3′MB-mediator resulted in significantly higher signals compared to 5′MB-labeled mediators. However, when using 5′MB-labeled mediators with a C3-blocking group at their 3′-end, the signal was comparable with those of 3′MB-labeled mediators (Figure 3). We thus conclude that the influence of the MB-label position on the signal is little, and both designs can be used. However, blocking the 3′-end of the mediator is highly recommended to avoid unspecific extension of the mediator, which could lead to stable duplexes that could not hybridize with the UR anymore. As MB already hinders extension, we favor the single modification mediator configuration with the 3′MB-label.

Figure 3.

Figure 3

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Position of the MB label is negligible as long as the 3′-end of the mediator is blocked (either with MB or a C3 group). Mediators with 5′MB-label without 3′C3 group are extensible. The lower signal indicates their involvement in unwanted reactions, which reduces their availability for hybridization with the UR. Depicted are the peak currents that were acquired with SWV each 20 s during the eMD LAMP of 104S. aureus (PTC) or no target DNA (NTC). The mediator concentration was 200 nM. Data points represent the mean and standard deviation of n = 3.

When analyzing the dependency of signal height and the ttp on mediator concentration, the combination of 200 nM mediator, 400 nM LoopMedc, and 400 nM Loop primer resulted in the highest absolute and relative signals (Figure 4 and Table S5). Lower mediator and LoopMedc primer concentrations resulted in lower signals and no significantly faster amplification. Higher mediator and LoopMedc primer concentrations increasingly affected the signal height and also the ttp. These findings were in line with the previously reported observations of reduced amplification efficiency for duplex primers in literature,29,46 even though we determined a slightly higher optimum concentration of mediator and LoopMedc concentration than in the fluorescence-based MD LAMP. This may be due to the different approach to determine the optimum. In the fluorescence approach, intercalating dyes were used. Our approach involved the signal generation reaction at the UR in the analysis. This may be more efficient at higher concentrations of released mediators and possibly compensates for the slightly affected amplification efficiency.

Figure 4.

Figure 4

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Best eMD LAMP signals were acquired for (200:400:400) nM mediator:LoopMedc:Loop primers. For higher mediator concentrations, the SWV peak currents and amplification speeds became increasingly affected. All PTC reactions contained 104 lysed S. aureus. Data points represent mean and standard deviation of n = 3.

Detection of MecA and T. pallidum with the Same UR

With the optimized reaction conditions, the LoD for the detection of the mecA gene from lysed S. aureus was determined. All reactions that contained 3 × 103 copies or more were reliably amplified. For 1 × 103 copies and 3 × 102 copies, 7/8 and 3/8 reactions became positive within the 30 min reaction time, respectively (Figure 5). Lower concentrations and NTC did not show a signal. The calculated 95% LoD was 1 × 103 copies per reaction (95% confidence interval (CI): 8 × 102–2 × 103 copies per reaction) or 40 aM referred to the reaction volume.

Figure 5.

Figure 5

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Real-time eMD LAMP reactions for the amplification of the mecA gene from crude S. aureus lysate conducted in Isothermal Amplification Buffer II with Bst 3.0. Eight, seven, and three out of eight reactions were positive for 3 × 103, 1 × 103, and 3 × 102 copies, respectively. The signals show the peak currents of SWV acquired each 20 s vs time.

The results were in line with the results of Nanayakkara and White, who also reported a LoD of 103 for mecA with the same primer set, but for genomic DNA instead of crude lysate and without LoopB- or LoopBMedc-primers, and with fluorogenic detection.32 For nucleic acid detection at universally functionalized electrodes, it is among the lowest LoDs requiring the shortest analysis time (Table S1).

Successful detection of spiked DNA in real swab samples demonstrates the diagnostic relevance of the approach. The tolerance against real samples was tested using self-swabbed samples from three individuals, which were eluted in a commercially available MSwab transport medium. All PTCs (Figure S4) show the expected signal increase, whereas all NTCs remained negative within the 30 min analysis time. Referred to the controls, reactions that contained eluate from nasal swab samples were slightly delayed by 1–7 min (Figure S4 and Table S6). However, even though the spiked MRSA concentration of 3.3 × 103 cells per reaction was close to the previously determined LoD, the presence of eluate from real nasal swab samples did not lead to false negatives. Equally important is that the NTCs show no indication of unspecific amplification. These results indicate the applicability of this approach for further clinical testing.

To demonstrate the universality of the approach, T. pallidum was amplified and detected using electrode arrays functionalized with the same UR sequence. Furthermore, amplification reactions were performed in different amplification buffers and with different polymerases to demonstrate a broader applicability. Reactions were conducted in triplicates.

Amplification was successful under all tested conditions (Figure 6). The fastest amplification was observed for Bst. 3.0 polymerase in Isothermal Amplification Buffer II, followed by Bst 2.0 warm-start polymerase in RM MPM buffer and then Bst 2.0 polymerase in Isothermal Amplification Buffer. The latter also exhibited the lowest electrochemical signal. However, the speed of amplification came at the cost of an increased risk of false positives. The observed signal behavior is comparable to previous experiments that compared Bst 3.0 with Bst 2.0.32 It indicates that electrochemical detection is suited for a wide range of amplification buffers and not just for specially optimized conditions. This should facilitate the development of new assays or the transfer of existing fluorescent assays.

Figure 6.

Figure 6

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Real-time eMD LAMP reactions for the amplification of T. pallidum in different amplification buffers. (A) Isothermal Amplification Buffer II with Bst 3.0 polymerase. (B) Isothermal Amplification Buffer with a Bst 2.0 warm-start polymerase. (C) RM MPM buffer with Bst 2.0 warm-start polymerase. The signals show the peak currents of SWV acquired each 20 s vs time.

The preliminary LoD for amplification in Isothermal Amplification Buffer II, RM MPM Buffer, and Isothermal Amplification Buffer was 4 × 102, 4 × 102, and 5 × 102 copies per reaction, respectively, if the cutoff time is chosen before the amplification of false-positives. This is in the same range as the reported LoD of 360 copies per reaction that Becherer et al. determined for a fluorescent MD LAMP reaction with RM MPM Buffer.9 However, more replicates and smaller dilution steps would be needed to determine the LoD more precisely (see the Supporting Information for detailed discussion).

Conclusions

Preparedness for the breakout of new diseases requires cost efficient consumables and flexible production lines that can be quickly repurposed according to the actual diagnostic demand.2,47

This motivated us to develop a concept for repurposable electrochemical nucleic acid detection by employing universally functionalized electrodes. The introduced eMD LAMP meets that demand for universal electrode functionalization without compromising sensitivity and ensuring selectivity by using probes for detection. Functionality and universality are demonstrated by real-time detection of S. aureus and T. pallidum amplification with electrodes that exhibit the same solid-phase probe sequence.

Compared with other strategies for universal electrochemical nucleic acid detection (Table S1), the combination of LAMP with mediator displacement leads to sensitive and remarkably faster results. In contrast to the only other current universal probe-based electrochemical real-time LAMP approach,28 our eMD LAMP is faster and requires only minimal adaptations to the established LAMP reaction. Apart from the solid-phase UR, only the addition of a mediator and the extension of one Loop primer by a mediator complementary sequence are required compared to the introduction of 3 additional oligonucleotides that are prehybridized to form a 3-way junction probe.28 This makes our method easily transferable to existing electrochemical platforms. The universal electrode functionalization allows easy adaptations to new targets, thereby saving time and costs for development, approval, and production. The demonstrated use of carbon electrodes and the covalent CMA-UR immobilization strategy will help to reduce costs and increase robustness under different reaction conditions37 when compared to thiol-modified gold electrodes.

The findings presented might help to make electrochemical molecular testing more cost-effective by using cheap materials (carbon electrodes) and enabling the large-scale production of a universal cartridge. Together with the repurposability of the cartridge, this should help to keep production lines operable also in interpandemic times and improve preparedness.

Acknowledgments

The authors gratefully acknowledge funding from the Ministry of Economic Affairs, Labour and Tourism Baden-Württemberg (Projects: 35-4223.10/10 and BW1_4156/02) and from the Federal Ministry for Economic Affairs and Climate Action (BMWK) on the basis of a decision by the German Bundestag (16KN078425). Z.B. acknowledges funding from the Alexander von Humboldt Foundation. We want to thank Quattrocento SAS (namely Mathieu Grisola and Damien Marchal) for enabling the rent of the Leo prototype and for the fruitful discussion, and Mast Diagnostica for providing the plasmid for the T. pallidum LAMP and RM MPM buffer. We want to thank Lisa Becherer for helpful discussions and assistance in designing the first LoopMedc primers.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.4c02492.

  • State-of-the-art for electrochemical nucleic acid detection with universally functionalized electrodes (Table S1); target independent oligonucleotides: universal reporters (UR) and mediators (Table S2); primer sequences for the detection of mecA gene of Staphylococcus aureus (Table S3); primer sequences for the detection of Treponema pallidum (Table S4); mediator concentration and the position of the methylene blue label influenced the signal height and time to positive (ttp) of the electrochemical MD LAMP (Table S5); time to positive for PTC reactions with nasal matrix of three different persons and for the respective controls (Table S6); the Leo electrochemical plate reader prototype of Easy Life Science/Quattrocento (Figure S1); data evaluation for electrochemical real-time MD-LAMP as conducted with the custom-made Python script (Figure S2); influence of LNA modifications in UR and/or Medc sequence on the signal tested by primer extension reaction (Figure S3); real swab samples of three test persons only lead to a slightly retarded signal increase compared to the control reactions (Figure S4); principle of loop-mediated isothermal amplification reaction (LAMP) as introduced by Notomi et al.10,11 (Scheme S1); principle of mediator displacement reactions and the expected signals (Scheme S2) (PDF)

Author Contributions

The manuscript was written through contributions of all authors.

The authors declare the following competing financial interest(s): The introduced eMD LAMP approach is partially protected by EP2017083039 and family. The patent is owned by the University of Freiburg and Hahn-Schickard. MT and FvS are co-inventors.

Supplementary Material

se4c02492_si_001.pdf (1.4MB, pdf)

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Supplementary Materials

se4c02492_si_001.pdf (1.4MB, pdf)

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