Colorimetric and electrochemical analysis of DNAzyme-LAMP amplicons for the detection of 
Food safety concerns around the globe are linked to Escherichia coli ( E. coli ) contamination in various food products , with non-pathogenic E. coli serving as a hygiene indicator in both food and water , . E. coli is commonly isolated from ready-to-eat products including fresh leafy vegetables – and non-pasteurized milk , posing a significant risk to consumers , as these undergo little to no antimicrobial treatment during processing . It is estimated that 600 million cases and 420,000 deaths are caused annually by foodborne gastroenteritis originating from fresh foods . Microbial contamination poses a significant threat to food safety, yet access to rapid and sensitive testing of food samples is limited. Microbial culture-based detection and conventional molecular methods, such as polymerase chain reaction (PCR), are the gold standard methods for detection and quantification of foodborne pathogens . However, inherent properties of these methods make them time-consuming and expensive, limiting their use at resource-limited testing sites. Loop-mediated isothermal amplification (LAMP), developed in 2000 , has been pursued as an alternative molecular method for the detection of microbial contaminants as it provides similar performance to PCR and culture while maintaining a rapid turnaround and simple equipment requirement. LAMP is highly specific , , using 4–6 primers to recognize 6–8 genomic loci to rapidly amplify target DNA under isothermal conditions. Further, LAMP possesses a unique tolerance to amplification inhibitors often present in food, clinical, and environmental samples , making it an attractive tool for microbial detection in food samples, which are often complex . While the LAMP amplification process itself has minimal equipment requirements, methods for sensitive and specific measurement of LAMP amplicons with minimal equipment are lacking. Fluorometers or turbidimeters have historically been used to monitor LAMP amplification – . However, equipment complexity limits their use as on-site diagnostic methods , . In contrast, the equipment for colorimetric and electrochemical readouts are relatively simple and accessible. Affordable and portable measurement options are commercially available for electrochemical and colorimetric readouts in smartphones and other hand-held devices. Public applications have been developed for smartphone-based analysis of colorimetric signals , and their use offers a portable alternative to bulky spectrophotometers in resource-limited settings . Electrochemical biosensors are easily miniaturized, enabling compatibility with fully automated microfluidic lab-on-a-chip designs – . Current detection methods designed for these readouts either require expensive reagents (modified primers , single-use electrodes – ) or monitor LAMP amplicon production indirectly. The latter senses the presence of double stranded DNA indiscriminately (intercalating dyes ) or amplification by-products (pH , ion accumulation – ), which can be sensitive to sample components . A recent approach, referred to herein as DNAzyme-LAMP, introduced a DNAzyme reporter into the LAMP amplicon itself as a simple means to specifically detect LAMP amplicons directly with a colorimetric , or electrochemical readout. In DNAzyme-LAMP, the DNAzyme complimentary sequence is incorporated into one , or both of the LAMP inner primers (forward or backward) such that the DNAzyme reporter is synthesized in each successful round of amplification . The DNAzyme reporter used in this approach, Dz-00, belongs to a specific class of DNAzymes containing a G-quadruplex motif that complexes with hemin to produce catalytic activity mimicking that of peroxidase enzymes . Such peroxidase-mimicking DNAzymes are able to catalyze the oxidation of many chromogenic peroxidase substrates, such as tetramethylbenzidine (TMB), to produce a colored radical ion that is detected by colorimetric , and electrochemical measurement methods. Our group recently developed a new method for electrochemical detection of TMB oxidation by a peroxidase enzyme, horseradish peroxidase (HRP), that was more sensitive than traditional TMB sensing methods. In this study, we adapted the new electrochemical TMB detection method to DNAzymes. In previous studies on electrochemical detection of oxidized TMB, the electrode surface was functionalizated with probes – for the oxidation reaction to take place at the electrode surface. This approach necessitates single-use electrodes which makes the testing expensive and difficult to assure reproducibility. On the other hand, our approach does not require functionalization of the electrode allowing for reuse of the microchip electrodes. Recently developed biosensors offered highly sensitive and specific detection of foodborne pathogens compared to traditional methods. However, the complexity of the T7 exonuclease-assisted PAM-free Dual CRISPR-Cas12a Biosensor , its multi-step procedure, and the need for a fluorescence reader may restrict its use in resource-limited settings. Additionally, while high-performance photo-active materials could enhance the sensitivity of the photoelectrochemical aptasensor for E. coli O157 detection , the complex preparation challenges the production of disposable electrodes for field use. Moreover, the use of Cadmium Sulfide (CdS) quantum dots, selected for their easy preparation and high conversion efficiency, raises toxicity concerns. These limitations impede the practical application of these sensors in resource-limited environments and hinder their widespread adoption. This study describes a novel approach for rapid and inexpensive detection of E. coli in food matrices by pairing an optimized DNAzyme-LAMP with traditional colorimetric measurement or our new electrochemical method. The performance of the optimized DNAzyme-LAMP for colorimetric detection of E. coli in food matrices was assessed in both leafy vegetables and milk. A novel method for electrochemical detection of DNAzyme activity is reported and coupled with DNAzyme-LAMP for a more sensitive alternative to colorimetric detection. This combination of optimized DNAzyme-LAMP with two different, highly catalytic DNAzymes, and the new electrochemical detection method improved the performance of DNAzyme-LAMP-based pathogen detection and distinguishes our study from previous DNAzyme-LAMP studies. Additionally, to our best knowledge, the incorporation of two different DNAzymes in LAMP amplification has not been explored in previous studies. The benchtop workflow for detecting E. coli from food matrices using the DNAzyme-LAMP approach is illustrated in Fig. . The workflow involves culture-free enrichment of E. coli from milk samples using magnetic beads functionalized with anti- E. coli polyclonal antibodies (Fig. a). For leafy vegetables, E. coli is recovered by washing the surface with water. This is followed by DNA extraction and DNAzyme-LAMP amplification using a heat block (Fig. b). On-site detection of the catalytic DNAzyme activity is performed either colorimetrically or electrochemically (Fig. c). The cumulative process (including immunomagnetic separation), achieves a sample-to-result time of less than 2 h.   Optimization of DNAzyme-LAMP reaction DNAzyme-LAMP is based on the amplification-dependent production of DNAzyme sequences, which is achieved through the addition of the DNAzyme reverse-complementary sequence (rcDNAzyme) into the inner primers (FIP and BIP). Whereas previous DNAzyme-LAMP reports have incorporated a single rcDNAzyme sequence, Dz-00, we designed inner primers containing various rcDNAzyme sequences. These primers, termed FIP-rc[DNAzyme] and BIP-rc[DNAzyme] (e.g. FIP-rcDz-00 ), are shown in Table (see methods) with the alignment of the LAMP primers on the target region of the E. coli phoA gene shown in Fig. a and Supplementary Fig. . Amplification of the target phoA gene region (Fig. b) results in catalytic LAMP amplicons which oxidize TMB, producing a product that is blue in color (Fig. c). Combinations of rcDNAzyme and control inner primers were systematically tested to identify a primer set with robust amplification and strong DNAzyme activity, which was measured as a specific colorimetric signal and represented by the signal-to-noise ratio (SNR) (Fig. a and Supplementary Fig. ). Primer sets containing FIP-rcEAD2 constantly produced strong DNAzyme activity and yielded stronger colorimetric signals than those utilizing other versions of FIP (FIP-rcDz00 and FIP-rcAGRO100) (Fig. a). As for BIP, BIP-rcDz00 and BIP-rcEAD2 containing sets both produced strong DNAzyme activity, with neither performing significantly better than the other (Fig. a). Collectively, this data suggests that the DNAzyme Dz-00 used in previous DNAzyme-LAMP studies is not a universally optimal DNAzyme for all rcDNAzyme primers. Overall, DNAzyme-LAMP reactions including two rcDNAzyme primers produced the strongest colorimetric signals (Fig. a). While AGRO100 primer sets underperformed colorimetrically with only one set, FIP-rcEAD2 & BIP-rcAGRO100 produced a positive signal above the NTC ( p = 0.033, one-tailed t-test) (Fig. a). The FIP-rcEAD2 & BIP-rcDz00 primer set consistently produced strong DNAzyme activity and thus was investigated for further assay development. Production of the corresponding DNAzyme-LAMP amplicon was confirmed with gel electrophoresis that showed an increase in amplicon size with rcDNAzyme primers indicating the production of DNAzyme sequences within the resulting amplicon (Supplementary Fig. ). Production of active DNAzyme sequences was confirmed by colorimetric reaction where DNAzyme-LAMP produced a strong signal that was not present in control LAMP (Supplementary Fig. ). Concentrations of inner (FIP and BIP) and loop (LF and LB) primers in the LAMP reaction were optimized to improve the assay analytical sensitivity (Fig. b) as well as LAMP amplification time (Supplementary Fig. ). An elevated concentration of primers (1.6 µM of FIP and BIP, and 0.6 µM of LF and LB) improved analytical sensitivity compared to the standard primer concentration commonly used in LAMP (0.8 µM FIP and BIP, and 0.4 µM of LF and LB) (Fig. b). Analytical specificity of DNAzyme-LAMP method for E. coli detection The DNAzyme-LAMP reaction produced strong and specific DNAzyme activity for all E. coli strains (reference strains and clinical isolates originating from various sample types) without cross-reaction to any of the non- E. coli bacteria that commonly contaminate food matrices, confirming the analytical specificity of the method was not affected by the addition of rcDNAzyme sequences (Fig. ). The colorimetric results aligned with gel electrophoresis, as amplicons showed the same characteristic banding-pattern of LAMP in all E. coli reference strains and clinical isolates (Supplementary Fig. ). The colorimetric signal produced by both E. coli clinical isolates and reference strains showed no significant difference (ANOVA comparison of group means, p = 0.89), indicating the assay’s potential for testing E. coli strains from various sources (Fig. b). Analytical sensitivity of colorimetric DNAzyme-LAMP method for E. coli detection Analytical sensitivity of the optimized colorimetric DNAzyme-LAMP reaction was established by extracting genomic DNA from serial dilutions of E. coli overnight culture. Using DNAzyme-LAMP and colorimetric measurement, an E. coli concentration of 1.8 log CFU (63.1 CFU)/reaction was consistently detected, with the assay limit of detection falling between 0.8 and 1.8 log CFU (6.3–63.1 CFU)/reaction (Fig. a). The analytical sensitivity of the DNAzyme-LAMP method was also evaluated using copies of the target phoA gene region produced by PCR. As shown in Fig. b, a minimum concentration of 0.5 aM gene copies/reaction was detected by the colorimetric DNAzyme-LAMP method, with the LOD between 0.1 and 0.5 aM target gene copies. Detection of E. coli in spiked food matrices by the DNAzyme-LAMP method The colorimetric DNAzyme-LAMP method was then evaluated for the detection of E. coli from the surface of leafy vegetables (lettuce, spinach, and cilantro) and milk. E. coli was recovered from spiked vegetable surfaces (8 log CFU/g) into a simple vegetable rinse medium. E. coli in the resulting vegetable rinse medium was detected with no significant differences between vegetable matrices (ANOVA, p = 0.875) (Fig. a). The colorimetric DNAzyme-LAMP was then tested with large-volume milk samples (10 mL). Milk was spiked with known amounts of E. coli (6.8, 4.8, and 2.8 log CFU/10 mL sample) and enriched by PAb-SMB prior to DNA preparation and DNAzyme-LAMP reaction. The recovery efficiency of PAb-SMB enrichment can be found in Supplementary Table . At each tested concentration, E. coli was recovered from the spiked milk sample and successfully detected by colorimetric DNAzyme-LAMP (Fig. b). To strengthen the validity, reliability, and overall performance of, we compared our novel colorimetric DNAzyme-LAMP assay against conventional PCR. E. coli in spiked vegetable rinse water and milk was detected both by DNAzyme-LAMP (Fig. ) and PCR (Supplementary Fig. ), supporting its practical applicability in routine diagnosis. Electrochemical detection of DNAzyme LAMP amplicons A method for electrochemical detection of DNAzyme-catalyzed TMB oxidation was developed for endpoint monitoring of the DNAzyme-LAMP reaction. We adapted our group’s previous approach for the detection of HRP enzyme activity to favor DNAzyme chemistry. Briefly, the ferrocyanide redox indicator was replaced with ferricyanide and the measurement buffer pH was elevated from acidic to alkaline. We first evaluated the ability of the electrochemical measurement to specifically sense the DNAzyme-LAMP amplicons. LAMP was carried out with the unmodified LAMP primer set to produce amplicons lacking DNAzyme sequences (Control amplicons). This control was tested alongside the DNAzyme-LAMP amplicon produced by rcDNAzyme primers (rcDNAzyme amplicon) as well as rcDNAzyme primers with no template DNA (no template control [NTC]). Sample preparation for electrochemical measurement was similar to that of colorimetric measurement in that amplicons were reacted with TMB; however, only 5 µL of amplicon was used in this reaction. The ferricyanide redox mediator was added following the TMB reaction, incubated, and the solution was transferred to a microchip for electrochemical measurement by a potentiostat. Nyquist plots resulting from the electrochemical measurement show clear differences between the impedance spectra of the rcDNAzyme amplicons and the control amplicons lacking active DNAzyme sequences (Fig. a). The DNAzyme-LAMP amplicons produced an expansion of both the reactive (Y-axis) and resistive (X-axis) impedance components compared to the NTC. This expansion was absent in the control amplicons. Instead, the spectra of the control amplicons overlap with the spectra produced by rcDNAzyme NTC. This data suggests that the novel electrochemical measurement method captured DNAzyme activity and was not impacted by other products of the LAMP amplification. Performance of electrochemical detection of DNAzyme-LAMP To assess the performance of the novel electrochemical detection method, the analytical sensitivity of DNAzyme-LAMP was evaluated using electrochemical measurements. Performance was first evaluated using genomic DNA templates extracted from serially diluted E. coli suspensions. Using the electrochemical detection method with DNAzyme-LAMP, the minimum bacterial concentration of 0.8 log CFU (6.3 CFU) per reaction was consistently detected (Fig. b). This concentration was not detected colorimetrically. Further, the average electrochemical signal (S/ N = 24.5) (Fig. b) was much stronger than the colorimetric signal (SNR = 6.4) (Fig. a). Analytical sensitivity was also evaluated using PCR-generated gene copies. Again, pairing electrochemical detection with DNAzyme-LAMP proved to be more sensitive than the colorimetric measurement with 0.1 aM gene copies reliably detected experimentally from purified culture (Fig. c) whereas 0.5 aM gene copies was the minimum detected colorimetrically (Fig. b). Together, this data suggests that the novel electrochemical detection method is more sensitive than colorimetric measurement.   DNAzyme-LAMP is based on the amplification-dependent production of DNAzyme sequences, which is achieved through the addition of the DNAzyme reverse-complementary sequence (rcDNAzyme) into the inner primers (FIP and BIP). Whereas previous DNAzyme-LAMP reports have incorporated a single rcDNAzyme sequence, Dz-00, we designed inner primers containing various rcDNAzyme sequences. These primers, termed FIP-rc[DNAzyme] and BIP-rc[DNAzyme] (e.g. FIP-rcDz-00 ), are shown in Table (see methods) with the alignment of the LAMP primers on the target region of the E. coli phoA gene shown in Fig. a and Supplementary Fig. . Amplification of the target phoA gene region (Fig. b) results in catalytic LAMP amplicons which oxidize TMB, producing a product that is blue in color (Fig. c). Combinations of rcDNAzyme and control inner primers were systematically tested to identify a primer set with robust amplification and strong DNAzyme activity, which was measured as a specific colorimetric signal and represented by the signal-to-noise ratio (SNR) (Fig. a and Supplementary Fig. ). Primer sets containing FIP-rcEAD2 constantly produced strong DNAzyme activity and yielded stronger colorimetric signals than those utilizing other versions of FIP (FIP-rcDz00 and FIP-rcAGRO100) (Fig. a). As for BIP, BIP-rcDz00 and BIP-rcEAD2 containing sets both produced strong DNAzyme activity, with neither performing significantly better than the other (Fig. a). Collectively, this data suggests that the DNAzyme Dz-00 used in previous DNAzyme-LAMP studies is not a universally optimal DNAzyme for all rcDNAzyme primers. Overall, DNAzyme-LAMP reactions including two rcDNAzyme primers produced the strongest colorimetric signals (Fig. a). While AGRO100 primer sets underperformed colorimetrically with only one set, FIP-rcEAD2 & BIP-rcAGRO100 produced a positive signal above the NTC ( p = 0.033, one-tailed t-test) (Fig. a). The FIP-rcEAD2 & BIP-rcDz00 primer set consistently produced strong DNAzyme activity and thus was investigated for further assay development. Production of the corresponding DNAzyme-LAMP amplicon was confirmed with gel electrophoresis that showed an increase in amplicon size with rcDNAzyme primers indicating the production of DNAzyme sequences within the resulting amplicon (Supplementary Fig. ). Production of active DNAzyme sequences was confirmed by colorimetric reaction where DNAzyme-LAMP produced a strong signal that was not present in control LAMP (Supplementary Fig. ). Concentrations of inner (FIP and BIP) and loop (LF and LB) primers in the LAMP reaction were optimized to improve the assay analytical sensitivity (Fig. b) as well as LAMP amplification time (Supplementary Fig. ). An elevated concentration of primers (1.6 µM of FIP and BIP, and 0.6 µM of LF and LB) improved analytical sensitivity compared to the standard primer concentration commonly used in LAMP (0.8 µM FIP and BIP, and 0.4 µM of LF and LB) (Fig. b).   E. coli detection The DNAzyme-LAMP reaction produced strong and specific DNAzyme activity for all E. coli strains (reference strains and clinical isolates originating from various sample types) without cross-reaction to any of the non- E. coli bacteria that commonly contaminate food matrices, confirming the analytical specificity of the method was not affected by the addition of rcDNAzyme sequences (Fig. ). The colorimetric results aligned with gel electrophoresis, as amplicons showed the same characteristic banding-pattern of LAMP in all E. coli reference strains and clinical isolates (Supplementary Fig. ). The colorimetric signal produced by both E. coli clinical isolates and reference strains showed no significant difference (ANOVA comparison of group means, p = 0.89), indicating the assay’s potential for testing E. coli strains from various sources (Fig. b).   E. coli detection Analytical sensitivity of the optimized colorimetric DNAzyme-LAMP reaction was established by extracting genomic DNA from serial dilutions of E. coli overnight culture. Using DNAzyme-LAMP and colorimetric measurement, an E. coli concentration of 1.8 log CFU (63.1 CFU)/reaction was consistently detected, with the assay limit of detection falling between 0.8 and 1.8 log CFU (6.3–63.1 CFU)/reaction (Fig. a). The analytical sensitivity of the DNAzyme-LAMP method was also evaluated using copies of the target phoA gene region produced by PCR. As shown in Fig. b, a minimum concentration of 0.5 aM gene copies/reaction was detected by the colorimetric DNAzyme-LAMP method, with the LOD between 0.1 and 0.5 aM target gene copies.   E. coli in spiked food matrices by the DNAzyme-LAMP method The colorimetric DNAzyme-LAMP method was then evaluated for the detection of E. coli from the surface of leafy vegetables (lettuce, spinach, and cilantro) and milk. E. coli was recovered from spiked vegetable surfaces (8 log CFU/g) into a simple vegetable rinse medium. E. coli in the resulting vegetable rinse medium was detected with no significant differences between vegetable matrices (ANOVA, p = 0.875) (Fig. a). The colorimetric DNAzyme-LAMP was then tested with large-volume milk samples (10 mL). Milk was spiked with known amounts of E. coli (6.8, 4.8, and 2.8 log CFU/10 mL sample) and enriched by PAb-SMB prior to DNA preparation and DNAzyme-LAMP reaction. The recovery efficiency of PAb-SMB enrichment can be found in Supplementary Table . At each tested concentration, E. coli was recovered from the spiked milk sample and successfully detected by colorimetric DNAzyme-LAMP (Fig. b). To strengthen the validity, reliability, and overall performance of, we compared our novel colorimetric DNAzyme-LAMP assay against conventional PCR. E. coli in spiked vegetable rinse water and milk was detected both by DNAzyme-LAMP (Fig. ) and PCR (Supplementary Fig. ), supporting its practical applicability in routine diagnosis.   A method for electrochemical detection of DNAzyme-catalyzed TMB oxidation was developed for endpoint monitoring of the DNAzyme-LAMP reaction. We adapted our group’s previous approach for the detection of HRP enzyme activity to favor DNAzyme chemistry. Briefly, the ferrocyanide redox indicator was replaced with ferricyanide and the measurement buffer pH was elevated from acidic to alkaline. We first evaluated the ability of the electrochemical measurement to specifically sense the DNAzyme-LAMP amplicons. LAMP was carried out with the unmodified LAMP primer set to produce amplicons lacking DNAzyme sequences (Control amplicons). This control was tested alongside the DNAzyme-LAMP amplicon produced by rcDNAzyme primers (rcDNAzyme amplicon) as well as rcDNAzyme primers with no template DNA (no template control [NTC]). Sample preparation for electrochemical measurement was similar to that of colorimetric measurement in that amplicons were reacted with TMB; however, only 5 µL of amplicon was used in this reaction. The ferricyanide redox mediator was added following the TMB reaction, incubated, and the solution was transferred to a microchip for electrochemical measurement by a potentiostat. Nyquist plots resulting from the electrochemical measurement show clear differences between the impedance spectra of the rcDNAzyme amplicons and the control amplicons lacking active DNAzyme sequences (Fig. a). The DNAzyme-LAMP amplicons produced an expansion of both the reactive (Y-axis) and resistive (X-axis) impedance components compared to the NTC. This expansion was absent in the control amplicons. Instead, the spectra of the control amplicons overlap with the spectra produced by rcDNAzyme NTC. This data suggests that the novel electrochemical measurement method captured DNAzyme activity and was not impacted by other products of the LAMP amplification.   To assess the performance of the novel electrochemical detection method, the analytical sensitivity of DNAzyme-LAMP was evaluated using electrochemical measurements. Performance was first evaluated using genomic DNA templates extracted from serially diluted E. coli suspensions. Using the electrochemical detection method with DNAzyme-LAMP, the minimum bacterial concentration of 0.8 log CFU (6.3 CFU) per reaction was consistently detected (Fig. b). This concentration was not detected colorimetrically. Further, the average electrochemical signal (S/ N = 24.5) (Fig. b) was much stronger than the colorimetric signal (SNR = 6.4) (Fig. a). Analytical sensitivity was also evaluated using PCR-generated gene copies. Again, pairing electrochemical detection with DNAzyme-LAMP proved to be more sensitive than the colorimetric measurement with 0.1 aM gene copies reliably detected experimentally from purified culture (Fig. c) whereas 0.5 aM gene copies was the minimum detected colorimetrically (Fig. b). Together, this data suggests that the novel electrochemical detection method is more sensitive than colorimetric measurement.   Identification of microbial food contamination during food production and processing is important for global food safety. LAMP has become a prominent technology in the development of sensitive molecular methods for rapid detection of microbial food contamination. The amplification process itself has minimal requirements. However, methods for specific measurement of LAMP amplicons with simple equipment requirements are lacking. We report a novel DNAzyme-LAMP detection system for rapid and inexpensive detection of E. coli in food matrices. The unique features of the described DNAzyme-LAMP system include the integration of two types of DNAzyme sequences (EAD2 and Dz-00) into a single amplicon without chemical modification of the primers. Also, we developed a highly sensitive electrochemical detection method that does not require electrode modifications. The developed DNAzyme-LAMP was compatible with food matrices and able to detect E. coli in both spiked vegetable and milk samples. The reported DNAzyme-LAMP is highly sensitive, with a colorimetric LOD between 6.3 and 63 CFU per reaction and 0.1–0.5 aM gene copies. The reported electrochemical detection method was more sensitive than colorimetric detection, with an LOD below 6.3 CFU per reaction and 0.1 aM gene copies, while also producing greater separation between positive and negative samples. Our method demonstrated superior analytical sensitivity compared to the 2.8 log CFU/g reported in prior research using real-time monitoring of fluorescence signals . Furthermore, the colorimetric and electrochemical readouts of our DNAzyme-LAMP method offer practical advantages over the fluorescence-based methods, facilitating easier and quicker interpretation of results without the need for complex instrumentation. The comparison of our method with previous LAMP methods for E. coli detection is presented in Supplementary Table . The previous reports of DNAzyme-LAMP implemented Dz-00 as the DNAzyme reporter in a 4-primer system, omitting loop primers from the LAMP reaction , . G-rich sequences, like those of peroxidase-mimicking DNAzymes, reduce the LAMP amplification efficiency . A previous DNAzyme-LAMP report mitigated this effect by introducing chemical modifications into the inner primers to increase the assay sensitivity. However, these modifications are expensive. Loop primers are known to substantially increase LAMP amplification efficiency by facilitating various steps of the reaction at a fraction of the cost , . Additionally, peroxidase-mimicking DNAzymes with higher catalytic activity than Dz-00 have been reported, with AGRO100 and EAD2 possessing some of the highest catalytic activities in the peroxidase-mimicking DNAzyme repertoire , . In this study, we tested the inclusion of loop primers and other DNAzyme candidates (AGRO100 and EAD2), which resulted in efficient amplification reactions and strong catalytic activity without the additional reagent costs associated with chemical primer modification. Additionally, amplicons containing EAD2 (produced by a single rc-EAD2 primer) possessed higher catalytic activities than those containing Dz-00 (produced by an rc-Dz00 primer). The analytical sensitivity and SNR were improved in our new DNAzyme-LAMP design compared to those reported previously , allowing for confident discrimination between positive and negative samples. The comparison of our method with previous DNAzyme-LAMP detection methods is presented in Supplementary Table . Thus far, only one study incorporated rcDNAzyme sequence in two LAMP primers, achieving an LOD of 1.37 aM SARS-CoV-2 gene copies using a colorimetric smartphone measurement . The previous design incorporated two of the same DNAzyme sequences (Dz-00) into amplicons. The four-primer LAMP reaction omitted the loop primers and included chemically modified inner primers (phosphorothioate FIP and BIP) carrying the rcDNAzyme sequence. Compared to the previous report , our DNAzyme-LAMP method using a 6-primer LAMP reaction to incorporate two different DNAzyme sequences (Dz-00 and EAD2) achieved greater analytical sensitivity without the added expense of chemical primer modification, detecting 0.5 aM gene copies colorimetrically and 0.1 aM gene copies electrochemically. It is expected that the colorimetric DNAzyme-LAMP described would be compatible with any smartphone-based measurement system able to measure relevant wavelengths of light (450 or 655 nm). TMB as a colorimetric substrate is commonly used in ELISA assays, and smartphone-based measurement platforms have been developed specifically for TMB measurement , , including image processing applications that are publicly available in app stores (e.g. SpotXel) . The described electrochemical detection method is based on our group’s previous approach which utilized a single redox mediator, potassium ferrocyanide, to detect TMB oxidation. The newly described method instead uses potassium ferricyanide as the redox mediator to transduce the signal readout. The electrochemical detection itself is simple with minimal data analysis, can be carried out with a simple potentiostat, and allows for the reuse of the microchip electrodes. DNA amplification schemes (RCA , EXPAR , enzyme-assisted target recycling , , , , PCR , and LAMP ) have been coupled with DNAzyme oxidation of TMB for electrochemical detection of DNA and small molecules. Most of the reported methods require the oxidation reaction to take place at the electrode surface, which is achieved through electrode functionalization with DNA molecules – . Methods reported with immobilized capture probes preclude simple reuse of electrodes and are less sensitive than our reported method with a minimum reported limit of detection of 57 fM , compared to 0.1 aM gene copies in our study. Functionalized electrodes are single use, expensive or difficult to regenerate and can only be used for a single target sequence while reusable electrodes minimize cost and waste per sample and provide the potential for multiplexing with the same electrode. Three schemes have been reported that couple DNA amplification (PCR , enzyme-assisted amplification , and LAMP ) with electrochemical detection of DNAzyme-catalyzed TMB in the bulk solution. These methods all use the same amperometric approach for electrochemical detection, measuring the current resulting from the interaction of oxidized TMB directly with the electrode. Our novel electrochemical detection method is conductometric, using a redox mediator to amplify the electrochemical signal produced by TMB oxidation. DNAzyme-LAMP paired with our conductometric electrochemical detection method provided a more sensitive detection in a shorter time than all three of the previous methods (PCR 5.0 × 10 2 CFU/mL in 2.5 h , enzyme assisted amplification 0.5aM in 2.6 h , and LAMP 6.5 CFU/ mL in 2 hrs ). Further, the prior LAMP-based electrochemical method reported an electrochemical LOD equivalent to the colorimetric LOD, while the electrochemical detection method described in this study was more sensitive than the colorimetric counterpart. Pathogenic E. coli is a common contaminant of both leafy vegetables – and milk , with non-pathogenic strains often serving as indicators or surrogate organisms to monitor food process hygiene , . DNAzyme-LAMP successfully detected low concentrations of non-pathogenic E. coli in both leafy vegetables and large-volume milk samples with a colorimetric readout. Isolating the target pathogen becomes increasingly challenging particularly if the enrichment period extends to 12–24 h for milk and lettuce, respectively, as recommended in the FDA Bacteriological Analytical Manual (BAM) protocol . Therefore, the implementation of immunomagnetic separation (IMS) proves to be an effective tool for enhancing E. coli recovery through selectively capturing and concentrating the target E. coli cells, thereby improving the overall accuracy of the DNAzyme-LAMP assay to successfully detect low concentrations of non-pathogenic E. coli in both leafy vegetables and large-volume milk samples with a colorimetric readout. However, a reduction in recovery rate to 38.8% (Supplementary Table ), similar to the previously reported 59.8% at higher E. coli cell densities, indicates that the capacity of the separation columns used in IMS was exceeded . This suggests a need for optimization to maintain efficiency and ensure reliable recovery rates at higher bacterial loads. In this study, we developed a new DNAzyme-LAMP system by using two different DNAzymes that possess high catalytic activities and by optimizing LAMP conditions. Additionally, we introduced a novel electrochemical detection method which allows recycling of microchip electrodes for reduced costs and waste. The new system is rapid (within 2 h) and highly sensitive, with an LOD of 0.1 aM gene copies. The DNAzyme-LAMP benchtop workflow has minimal equipment requirements and can be carried out in resource-limited settings if combined with one of the many publicly available colorimetric smartphone-based measurement platforms. Our study could serve as the basis for closed-detection systems, as each of the sample processing methods used in this study can be made compatible with microfluidic designs (DNA extraction , , magnetic beads , ), allowing the potential for future integration of the DNAzyme-LAMP system into a fully-automated microfluidic biosensor when combined with our novel electrochemical detection method. This closed system can effectively mitigate the risk of false positive results due to aerosol contamination in LAMP assays, ensuring the accuracy and reliability of their findings. To further reduce false positive reactions, we implemented stringent protocols, including the use of separate rooms for pathogen preparation, DNA extraction, and the LAMP reaction. Additionally, the use of a commercial LAMP reaction mixture, instead of individual reagents, further reduced false positives. These measures not only improve the current assay but also pave the way for developing advanced, closed-system biosensing technologies in the future. The reported DNAzyme-LAMP technology could serve as a base sensing approach for on-site detection not only of E. coli , but also other organisms, at resource limited food processing sites. This approach has some limitations, including the requirement for separate facilities and stringent protocols, which may not be practical in all settings. The use of a commercial LAMP mixture, while effective in reducing false positives, adds to costs and dependence on proprietary reagents, which could limit accessibility and scalability for broader use. Additionally, the relative inconsistency between replicates observed in our study could be attributed to poor amplification efficiency of LAMP at low cell levels, which is known to cause a significant increase in the standard deviation of low copy numbers compared to higher copy numbers . Similar issues with poor quantification at low cell levels have been reported previously in which one to two out of three repeats showed as positive , , .   Principle of rcDNAzyme LAMP In this study, a 6-primer system (F3, B3, FIP, BIP, loop F and loop B) was used for LAMP reactions. LAMP primers were selected for the amplification of the E. coli phoA gene based on a previous work . The primer sequences are shown in Table . All primers were synthesized and obtained from Integrated DNA Technologies (IDT, Coralville, IA). A schematic illustration of the DNAzyme-LAMP mode of action is outlined in Fig. . The proposed DNAzyme-LAMP is based on the amplification-dependent production of two DNAzyme sequences in each round of amplification. The LAMP inner primers (i.e., FIP and BIP) contain one of three different DNAzyme reverse-compliment sequences (AGRO100, Dz-00, and EAD2) , , (rcDNAzyme, italicized in Table ) as a linker sequence between the target hybridization sites. These primers are termed [primer]-rc[DNAzyme] (e.g. FIP-rcEAD2). DNAzymes are introduced into the growing amplicon as the rcDNAzyme inner primers are extended, resulting in exponential amplification of both DNAzyme sequences. The rcDNAzyme primers are expected to increase the length of the exponentially amplified region by 35 nucleotides compared to the original, unmodified primers (control primers). Bacterial culture and DNA extraction Escherichia coli strains include reference strains (K-12, ATCC43894, ATCC25922, and ATCC15597), previously reported clinical isolates (727, DG 02–5, AG 01–7, AG 01–9, AG 01–10, and AG01-8) provided by the Department of Animal Science, The University of Tennessee – , and two E. coli isolates from domestic canine (diagnosed with either canine pyoderma or urinary tract infection) that were identified by the Clinical Bacteriology and Mycology lab (Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee). Bacterial strains were cultured aerobically for 18 h at 37 °C in 5 mL of Trypticase Soy Broth (TSB Becton Dickinson, Franklin Lakes, NJ, USA) in a shaking incubator at 225 rpm. The bacterial overnight culture was diluted in Luria-Bertani broth to obtain an optical density (OD) of 1.0 at 600 nm, and viable cell count (CFU/mL) was assessed by spreading the 100 µL of inoculated culture on a Trypticase Soy Agar (TSA) plate. The culture suspension was pelleted by centrifugation at 14,000 rpm for 2 min, and genomic DNA was extracted using the Wizard ® Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. DNA concentration was determined by NanoDrop 2200c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and the extracted DNA was used as a template for LAMP assays. LAMP conditions The LAMP reaction was carried out as described below at the final reaction volume of 25 µL. A 10× primer stock solution (16 µM FIP and BIP, 2 µM F3 and B3, and 6 µM of LF and LB primers) was prepared in nuclease-free water. The LAMP reaction mixture contained WarmStart LAMP 2× Master Mix (12.5 µL) (New England Biolabs, Ipswich, MA, USA), 10× primer stock solution (2.5 µL), nuclease-free water (7.5 µL), and the genomic DNA sample (2.5 µL containing10ng/ µL unless otherwise stated). The reaction mixture was then incubated at 65 °C on a heating block for 40–60 min (Supplementary Fig. ), followed by inactivation of LAMP enzymes at 95 °C for 5 min. A reaction mixture without genomic DNA (water) was used as a negative control (No Template Control [NTC]). DNAzyme-LAMP products were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide (85 V, 45 min). DNA bands were visualized under UV light. Colorimetric assessment of DNAzyme activity Following heat inactivation, DNAzyme-LAMP product (20 µL) was cooled to room temperature for 5 min, followed by the addition of hemin (MP Biomedicals. Santa Ana, CA) suspension (1.35 µL of 200 µM in Dimethyl sulfoxide (DMSO) and the solution was incubated at room temperature for 20 min. The mixture (20 µL) was incubated with TMB solution (100 µL of prepared Pierce Tetramethylbenzidine Substrate Kit, ThermoFisher Scientific) at room temperature for time course measurement of 2–8 min. Optical density (OD) at 655 nm was measured using a plate reader Model 680 (Bio-Rad, Hercules, CA). Validation of DNAzyme-LAMP assay’s analytical specificity The analytical specificity of the unmodified LAMP primer set used in this study was previously confirmed with 58 bacterial strains . The analytical specificity of the rcDNAzyme primer set was assessed using DNA extracted from Salmonella cholera suis ATCC BAA 664 and Salmonella Typhimurium ATCC 14028 reference strains, as well as Pseudomonas aeruginosa and Staphylococcus aureus clinical isolates (provided by the Department of Animal Science, The University of Tennessee). DNAzyme-LAMP was also assessed for detection of the E. coli reference strains and clinical isolates described above. Analytical sensitivity of the DNAzyme LAMP for E. coli detection Genomic DNA templates were prepared from ten-fold serial dilutions of E. coli overnight culture (OD 600 nm = 1.0). DNA was extracted from aliquots (1 mL) of each dilution, as described above. Gene copy DNA templates were produced by PCR amplification of the phoA gene region of E. coli genomic DNA. The PCR reaction was carried out with solution (50 µL) containing 1× PowerUp SYBER Master Mix (Applied Biosystems, Waltham, MA), F3 and B3 LAMP primer (0.4 µM each), and E. coli genomic DNA (10 ng). For PCR, a thermal cycler (C1000, BioRad) was programmed as follows: 1 min initial denaturation at 95 °C followed by 40 cycles of denature (95 °C, 15 s), annealing (50 °C, 30s) and extension (60 °C, 1 min). The PCR products (273 bp) were then confirmed by 2% agarose gel electrophoresis and purified using E.Z.N.A. ® Gel Extraction Kit (Omega Bio-Tek. Norcross, GA, USA). Ten-fold serial dilutions of PCR products, starting at 10 fM, served as the gene template for DNAzyme-LAMP. Detection of E. coli DNA from spiked vegetable matrices Three types of leafy vegetables (romaine lettuce, spinach, and cilantro) were obtained from a retail store, and the samples were prepared as previously described , . Briefly, vegetable samples (1 g) were rinsed four times with sterile water, placed in a biosafety cabinet, and exposed to ultraviolet light for 10 min at room temperature. The samples were pre-screened for the presence of E. coli by culturing on MacConkey agar plates (ThermoFisher Scientific. Waltham, MA). Vegetable samples with negative culture results were used in spiking experiments, with non-spiked vegetables serving as negative controls. The vegetable samples were spiked with target bacteria as described previously and analyzed immediately. The vegetable sample (1 g) was submerged in 10mL of TSB medium with an estimated 10 8 CFU spiking concentration followed by thoroughly mixing at 80 rpm for one hour at room temperature to allow the attachment of the bacteria to the plant surface. The leaves were then placed into 10 mL of sterile TSB rinse medium, and the bacteria attached to the surface of the vegetable were dissociated into the medium by vigorous shaking at 115 rpm for 10 min at room temperature. DNA was extracted from the pelleted bacterial cells recovered in the vegetable rinse medium by centrifugation as described above, and aliquots (2.5 µL) of each DNA extract were used for downstream DNAzyme-LAMP, and PCR as described above. Enrichment of E. coli in milk samples by immunomagnetic separation Aliquots (10 mL each) of pasteurized whole milk purchased from the supermarket were spiked with bacteria suspension (10 2 , 10 4 , and 10 6 CFU/mL) . E. coli in milk was enriched by antibody-coated magnetic beads as follows. Briefly, 50 µL of streptavidin-coated magnetic beads (SMB, New England Biolabs, Ipswich, MA) were incubated with 5 µL of biotinylated anti- E. coli polyclonal antibody (PAb) (5 mg/mL in 0.1% sodium azide, Invitrogen, Carlsbad, CA) for 10 min at 37 °C. The PAb-coated SMBs (PAb-SMB) were added to the E. coli -spiked or non-spiked milk and kept at 37 °C for 30 min. The PAb-SMBs were collected by standing for 1 min. on a magnetic bead separation rack (ThermoFisher Scientific). Then, the PAb-SMBs were washed twice with 200 µL of PBST (10 mM PBS with 0.05 v/v% Tween 20, pH 7.2) and resuspended in 1 mL of PBST. Recovery of E. coli (Supplementary Table ) was determined by culturing 10 µL of PAb-SMBs on MacConkey agar plates as described above. The recovery rate was calculated as the percentage ratio of the measured concentration to the spiked concentration according to [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\:Recovery\:rate\:(\%)=()\:100$$ . The remaining PAb-SMBs were used for DNA extraction using Qiagen DNeasy Tissue kit following the manufacturer’s instructions (Qiagen, Germantown MD). Aliquots (2.5 µL) of each DNA extract were used for downstream DNAzyme-LAMP, and PCR as described above. Electrode cleaning and preparation Dropsens (Asturias, Spain) IDEAU-200 gold interdigitated microchip electrodes (Au-IDEs) with ceramic substrates served as the working electrode in all experiments. Electrodes were cleaned in an acetone bath for 15 min. Upon removal, electrodes were rinsed briefly with acetone, isopropyl alcohol (IPA), and water sequentially. Each was allowed to flow over the electrode for 5 s before being air dried (15s). Electrodes underwent the above rinsing process (30 s) between sample measurements. Electrochemical measurement All scans were performed using a two-electrode system (Au-IDE) comprised of a working and counter electrode with an Autolab potentiostat (Metrohm, Riverview, FL) using the manufacturer’s FRA32M impedance module within a 200 Hz to 0.1 Hz scan range and a 20 mV rms excitation potential. A sample volume of 10 µL was used for all measurements. An initial electrochemical impedance spectroscopy (EIS) was performed with a 25 mM potassium ferricyanide K 3 [Fe(CN) 6 ] and 25 mM potassium ferrocyanide K 4 [Fe(CN) 6 ] solution in water on a cleaned, Au-IDE to ensure chip integrity. Electrochemical detection of amplicons is a modification of the method previously reported by our group . DNAzyme-LAMP amplicons (5 µL) were incubated with hemin (as described above) and mixed with 5 µL of freshly prepared TMB solution. The TMB reaction was allowed to proceed for 6 min. The solution was mixed with 2 µL of 25 mM K 3 [Fe(CN) 6 ] in water and incubated for an additional 15 min. After the reaction, 10 µL sample was transferred onto the Au-IDE for immediate impedance measurement, and electrochemical detection. Simultaneously, another aliquot from the same tube was incubated with hemin, then mixed with a freshly prepared TMB solution, and the reaction was detected colorimetrically (as described above). The same microchip electrode was used throughout each individual experiment and was rinsed briefly (described above) between samples. Data was recorded and analyzed using Autolab’s NOVA software. Data and statistics Graphical outputs were generated by GraphPad Prism software (version 8; GraphPad Software Inc.). Individual data points represent a single replicate in an experiment. Statistics carried out in R studio (version 2023.03.0 + 386) and GraphPad Prism (version 8). Multiple comparisons of groups (treatment effects) assessed using one way ANOVA with Dunnett’s post-hoc analysis. Single comparisons of Sample to NTC were performed by unpaired t-test. Signal-to-noise ratio (SNR or S/N), defined as the ratio of positive signal to that of the true negative, was calculated for each experimental replicate. SNR was calculated for colorimetric data according to Eq. and S/N was calculated for electrochemical data according to Eq. . 1 [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\:SNR=\:_{Sample}-\:{OD}_{Blank}}{{OD}_{NTC\:}-\:{OD}_{Blank}}$$ 2 [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\:S/N=\:_{Sample}}{{Z}_{NTC\:}}$$ Where NTC refers to no template control LAMP reaction, blank refers to buffer background, OD refers to optical density at 655 nm, and Z refers to the imaginary impedance component, -Z”, at 0.1 Hz. The limit of detection was calculated according to 3σ as described previously . Briefly, the positive cut off value was calculated as the mean + 3σ of SNR values obtained with triplicate NTC samples. The LOD was defined as the most dilute sample whose signal (OD or -Z”) was above the cut off and significantly greater than the NTC.   In this study, a 6-primer system (F3, B3, FIP, BIP, loop F and loop B) was used for LAMP reactions. LAMP primers were selected for the amplification of the E. coli phoA gene based on a previous work . The primer sequences are shown in Table . All primers were synthesized and obtained from Integrated DNA Technologies (IDT, Coralville, IA). A schematic illustration of the DNAzyme-LAMP mode of action is outlined in Fig. . The proposed DNAzyme-LAMP is based on the amplification-dependent production of two DNAzyme sequences in each round of amplification. The LAMP inner primers (i.e., FIP and BIP) contain one of three different DNAzyme reverse-compliment sequences (AGRO100, Dz-00, and EAD2) , , (rcDNAzyme, italicized in Table ) as a linker sequence between the target hybridization sites. These primers are termed [primer]-rc[DNAzyme] (e.g. FIP-rcEAD2). DNAzymes are introduced into the growing amplicon as the rcDNAzyme inner primers are extended, resulting in exponential amplification of both DNAzyme sequences. The rcDNAzyme primers are expected to increase the length of the exponentially amplified region by 35 nucleotides compared to the original, unmodified primers (control primers).   Escherichia coli strains include reference strains (K-12, ATCC43894, ATCC25922, and ATCC15597), previously reported clinical isolates (727, DG 02–5, AG 01–7, AG 01–9, AG 01–10, and AG01-8) provided by the Department of Animal Science, The University of Tennessee – , and two E. coli isolates from domestic canine (diagnosed with either canine pyoderma or urinary tract infection) that were identified by the Clinical Bacteriology and Mycology lab (Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee). Bacterial strains were cultured aerobically for 18 h at 37 °C in 5 mL of Trypticase Soy Broth (TSB Becton Dickinson, Franklin Lakes, NJ, USA) in a shaking incubator at 225 rpm. The bacterial overnight culture was diluted in Luria-Bertani broth to obtain an optical density (OD) of 1.0 at 600 nm, and viable cell count (CFU/mL) was assessed by spreading the 100 µL of inoculated culture on a Trypticase Soy Agar (TSA) plate. The culture suspension was pelleted by centrifugation at 14,000 rpm for 2 min, and genomic DNA was extracted using the Wizard ® Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. DNA concentration was determined by NanoDrop 2200c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and the extracted DNA was used as a template for LAMP assays.   The LAMP reaction was carried out as described below at the final reaction volume of 25 µL. A 10× primer stock solution (16 µM FIP and BIP, 2 µM F3 and B3, and 6 µM of LF and LB primers) was prepared in nuclease-free water. The LAMP reaction mixture contained WarmStart LAMP 2× Master Mix (12.5 µL) (New England Biolabs, Ipswich, MA, USA), 10× primer stock solution (2.5 µL), nuclease-free water (7.5 µL), and the genomic DNA sample (2.5 µL containing10ng/ µL unless otherwise stated). The reaction mixture was then incubated at 65 °C on a heating block for 40–60 min (Supplementary Fig. ), followed by inactivation of LAMP enzymes at 95 °C for 5 min. A reaction mixture without genomic DNA (water) was used as a negative control (No Template Control [NTC]). DNAzyme-LAMP products were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide (85 V, 45 min). DNA bands were visualized under UV light.   Following heat inactivation, DNAzyme-LAMP product (20 µL) was cooled to room temperature for 5 min, followed by the addition of hemin (MP Biomedicals. Santa Ana, CA) suspension (1.35 µL of 200 µM in Dimethyl sulfoxide (DMSO) and the solution was incubated at room temperature for 20 min. The mixture (20 µL) was incubated with TMB solution (100 µL of prepared Pierce Tetramethylbenzidine Substrate Kit, ThermoFisher Scientific) at room temperature for time course measurement of 2–8 min. Optical density (OD) at 655 nm was measured using a plate reader Model 680 (Bio-Rad, Hercules, CA).   The analytical specificity of the unmodified LAMP primer set used in this study was previously confirmed with 58 bacterial strains . The analytical specificity of the rcDNAzyme primer set was assessed using DNA extracted from Salmonella cholera suis ATCC BAA 664 and Salmonella Typhimurium ATCC 14028 reference strains, as well as Pseudomonas aeruginosa and Staphylococcus aureus clinical isolates (provided by the Department of Animal Science, The University of Tennessee). DNAzyme-LAMP was also assessed for detection of the E. coli reference strains and clinical isolates described above.   E. coli detection Genomic DNA templates were prepared from ten-fold serial dilutions of E. coli overnight culture (OD 600 nm = 1.0). DNA was extracted from aliquots (1 mL) of each dilution, as described above. Gene copy DNA templates were produced by PCR amplification of the phoA gene region of E. coli genomic DNA. The PCR reaction was carried out with solution (50 µL) containing 1× PowerUp SYBER Master Mix (Applied Biosystems, Waltham, MA), F3 and B3 LAMP primer (0.4 µM each), and E. coli genomic DNA (10 ng). For PCR, a thermal cycler (C1000, BioRad) was programmed as follows: 1 min initial denaturation at 95 °C followed by 40 cycles of denature (95 °C, 15 s), annealing (50 °C, 30s) and extension (60 °C, 1 min). The PCR products (273 bp) were then confirmed by 2% agarose gel electrophoresis and purified using E.Z.N.A. ® Gel Extraction Kit (Omega Bio-Tek. Norcross, GA, USA). Ten-fold serial dilutions of PCR products, starting at 10 fM, served as the gene template for DNAzyme-LAMP.   E. coli DNA from spiked vegetable matrices Three types of leafy vegetables (romaine lettuce, spinach, and cilantro) were obtained from a retail store, and the samples were prepared as previously described , . Briefly, vegetable samples (1 g) were rinsed four times with sterile water, placed in a biosafety cabinet, and exposed to ultraviolet light for 10 min at room temperature. The samples were pre-screened for the presence of E. coli by culturing on MacConkey agar plates (ThermoFisher Scientific. Waltham, MA). Vegetable samples with negative culture results were used in spiking experiments, with non-spiked vegetables serving as negative controls. The vegetable samples were spiked with target bacteria as described previously and analyzed immediately. The vegetable sample (1 g) was submerged in 10mL of TSB medium with an estimated 10 8 CFU spiking concentration followed by thoroughly mixing at 80 rpm for one hour at room temperature to allow the attachment of the bacteria to the plant surface. The leaves were then placed into 10 mL of sterile TSB rinse medium, and the bacteria attached to the surface of the vegetable were dissociated into the medium by vigorous shaking at 115 rpm for 10 min at room temperature. DNA was extracted from the pelleted bacterial cells recovered in the vegetable rinse medium by centrifugation as described above, and aliquots (2.5 µL) of each DNA extract were used for downstream DNAzyme-LAMP, and PCR as described above.   E. coli in milk samples by immunomagnetic separation Aliquots (10 mL each) of pasteurized whole milk purchased from the supermarket were spiked with bacteria suspension (10 2 , 10 4 , and 10 6 CFU/mL) . E. coli in milk was enriched by antibody-coated magnetic beads as follows. Briefly, 50 µL of streptavidin-coated magnetic beads (SMB, New England Biolabs, Ipswich, MA) were incubated with 5 µL of biotinylated anti- E. coli polyclonal antibody (PAb) (5 mg/mL in 0.1% sodium azide, Invitrogen, Carlsbad, CA) for 10 min at 37 °C. The PAb-coated SMBs (PAb-SMB) were added to the E. coli -spiked or non-spiked milk and kept at 37 °C for 30 min. The PAb-SMBs were collected by standing for 1 min. on a magnetic bead separation rack (ThermoFisher Scientific). Then, the PAb-SMBs were washed twice with 200 µL of PBST (10 mM PBS with 0.05 v/v% Tween 20, pH 7.2) and resuspended in 1 mL of PBST. Recovery of E. coli (Supplementary Table ) was determined by culturing 10 µL of PAb-SMBs on MacConkey agar plates as described above. The recovery rate was calculated as the percentage ratio of the measured concentration to the spiked concentration according to [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\:Recovery\:rate\:(\%)=()\:100$$ . The remaining PAb-SMBs were used for DNA extraction using Qiagen DNeasy Tissue kit following the manufacturer’s instructions (Qiagen, Germantown MD). Aliquots (2.5 µL) of each DNA extract were used for downstream DNAzyme-LAMP, and PCR as described above.   Dropsens (Asturias, Spain) IDEAU-200 gold interdigitated microchip electrodes (Au-IDEs) with ceramic substrates served as the working electrode in all experiments. Electrodes were cleaned in an acetone bath for 15 min. Upon removal, electrodes were rinsed briefly with acetone, isopropyl alcohol (IPA), and water sequentially. Each was allowed to flow over the electrode for 5 s before being air dried (15s). Electrodes underwent the above rinsing process (30 s) between sample measurements.   All scans were performed using a two-electrode system (Au-IDE) comprised of a working and counter electrode with an Autolab potentiostat (Metrohm, Riverview, FL) using the manufacturer’s FRA32M impedance module within a 200 Hz to 0.1 Hz scan range and a 20 mV rms excitation potential. A sample volume of 10 µL was used for all measurements. An initial electrochemical impedance spectroscopy (EIS) was performed with a 25 mM potassium ferricyanide K 3 [Fe(CN) 6 ] and 25 mM potassium ferrocyanide K 4 [Fe(CN) 6 ] solution in water on a cleaned, Au-IDE to ensure chip integrity. Electrochemical detection of amplicons is a modification of the method previously reported by our group . DNAzyme-LAMP amplicons (5 µL) were incubated with hemin (as described above) and mixed with 5 µL of freshly prepared TMB solution. The TMB reaction was allowed to proceed for 6 min. The solution was mixed with 2 µL of 25 mM K 3 [Fe(CN) 6 ] in water and incubated for an additional 15 min. After the reaction, 10 µL sample was transferred onto the Au-IDE for immediate impedance measurement, and electrochemical detection. Simultaneously, another aliquot from the same tube was incubated with hemin, then mixed with a freshly prepared TMB solution, and the reaction was detected colorimetrically (as described above). The same microchip electrode was used throughout each individual experiment and was rinsed briefly (described above) between samples. Data was recorded and analyzed using Autolab’s NOVA software.   Graphical outputs were generated by GraphPad Prism software (version 8; GraphPad Software Inc.). Individual data points represent a single replicate in an experiment. Statistics carried out in R studio (version 2023.03.0 + 386) and GraphPad Prism (version 8). Multiple comparisons of groups (treatment effects) assessed using one way ANOVA with Dunnett’s post-hoc analysis. Single comparisons of Sample to NTC were performed by unpaired t-test. Signal-to-noise ratio (SNR or S/N), defined as the ratio of positive signal to that of the true negative, was calculated for each experimental replicate. SNR was calculated for colorimetric data according to Eq. and S/N was calculated for electrochemical data according to Eq. . 1 [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\:SNR=\:_{Sample}-\:{OD}_{Blank}}{{OD}_{NTC\:}-\:{OD}_{Blank}}$$ 2 [12pt]{minimal} 				 				  				  				  				 				 				 				 				$$\:S/N=\:_{Sample}}{{Z}_{NTC\:}}$$ Where NTC refers to no template control LAMP reaction, blank refers to buffer background, OD refers to optical density at 655 nm, and Z refers to the imaginary impedance component, -Z”, at 0.1 Hz. The limit of detection was calculated according to 3σ as described previously . Briefly, the positive cut off value was calculated as the mean + 3σ of SNR values obtained with triplicate NTC samples. The LOD was defined as the most dilute sample whose signal (OD or -Z”) was above the cut off and significantly greater than the NTC.   Below is the link to the electronic supplementary material. Supplementary Material 1   