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2.
Anal Biochem ; 598: 113705, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32246925

RESUMO

Genosensors for the detection of DNA via hybridisation normally require post-amplification processing such as the generation of single-stranded DNA and pre-detection labelling, complicating and lengthening the assay. A straightforward electrochemical genosensor, for the direct detection of isothermally generated nucleic acid amplicons via hybridisation is reported. The detection of Karlodinium armiger, responsible for harmful algae blooms was used as a model system to demonstrate the proof of concept. The approach exploits the use of specifically modified primers designed to generate amplicons with a central duplex flanked by a single-stranded tail at one end of the duplex and a horse-radish peroxidase on the other end. Individual gold electrodes of an array were functionalised with self-assembled monolayers of short thiolated DNA probes, designed to hybridise with the single-stranded tailed amplicon with the reporter enzyme label incorporated. The optimum amplification time was determined to be 60 min, at a fixed temperature of 37 °C. The hybridisation time to the enzyme labelled amplicon was optimised to be 10 min, but 2 min hybridisation time was also adequate. In this first example of using horse radish peroxidase-labelled primer in solution-phase recombinase polymerase amplification for subsequent detection via solid-phase hybridisation, the detection limit achieved was 0.4 fM, equivalent to 27622 cells/L, and the developed genosensor was applied to the detection of synthetic as well as genomic DNA, which had been extracted from a seawater sample.

3.
Anal Chim Acta ; 1112: 54-61, 2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32334682

RESUMO

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

4.
Anal Chem ; 92(7): 4858-4865, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32133843

RESUMO

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 µg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

5.
Chemistry ; 26(6): 1286-1291, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31725178

RESUMO

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.


Assuntos
DNA/química , Compostos Ferrosos/química , Metalocenos/química , Nucleotídeos/química , Coloração e Rotulagem/métodos , Citidina Trifosfato/química , DNA/metabolismo , Sondas de DNA/síntese química , Sondas de DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Técnicas Eletroquímicas , Oxirredução , Especificidade por Substrato
6.
Talanta ; 207: 120308, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594570

RESUMO

Given the threat that ostreid herpesvirus 1 (OsHV-1) poses to shellfish aquaculture, the need for rapid, user-friendly and cost-effective methods to detect this marine pathogen and minimise its impact is evident. In this work, an electrochemical biosensor for the detection of OsHV-1 based on isothermal recombinase polymerase amplification (RPA) was developed. The system was first tested and optimised on maleimide microtitre plates as a proof-of-concept, before being implemented on miniaturised gold electrodes. Amperometric detection of the isothermally amplified product was achieved through a sandwich hybridisation assay with an immobilised thiolated capture probe and a horseradish peroxidase (HRP)-labelled reporter probe. Calibration curves were constructed using PCR-amplified OsHV-1 DNA, achieving a limit of detection of 207 OsHV-1 target copies. The biosensor was applied to the analysis of 16 oyster samples from an infectivity experiment, and results were compared with those obtained by qPCR analysis, showing a strong degree of correlation (r = 0.988). The simplicity, rapidity, cost-effectiveness and potential for in-situ testing with the developed biosensor provide a valuable tool for the detection of OsHV-1 in aquaculture facilities, improving their management.


Assuntos
Técnicas Biossensoriais/métodos , Crassostrea/virologia , Vírus de DNA/genética , DNA Viral/análise , DNA Viral/genética , Miniaturização , Temperatura , Animais , Técnicas Biossensoriais/economia , Calibragem , Calorimetria , Análise Custo-Benefício , Eletroquímica , Eletrodos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Fatores de Tempo
7.
ACS Omega ; 4(23): 20188-20196, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31815219

RESUMO

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.

8.
Trends Biotechnol ; 37(12): 1278-1281, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31399265

RESUMO

The use of isothermal nucleic acid amplification strategies to detect harmful algal blooms (HABs) is in its infancy. We describe recent advances in these systems and highlight the challenges for the achievement of simple, low-cost, compact, and portable devices for field applications.

9.
Sci Total Environ ; 689: 655-661, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279212

RESUMO

Ostreopsis cf. ovata is a benthic microalga distributed in tropical and temperate regions worldwide which produces palytoxins (PlTXs). Herein, an electrochemical biosensor for the detection of this toxic microalga is described. The detection strategy involves isothermal recombinase polymerase amplification (RPA) of the target using tailed primers and a sandwich hybridisation assay on maleimide-coated magnetic beads immobilised on electrode arrays. The biosensor attained a limit of detection of 9 pg/µL of O. cf. ovata DNA (which corresponds to ~640 cells/L), with no interferences from two non-target Ostreopsis species (O. cf. siamensis and O. fattorussoi). The biosensor was applied to the analysis of planktonic and benthic environmental samples. Electrochemical O. cf. ovata DNA quantifications demonstrated an excellent correlation with other molecular methods (qPCR and colorimetric assays) and allowed the construction of a predictive regression model to estimate O. cf. ovata cell abundances. This new technology offer great potential to improve research, monitoring and management of O. cf. ovata and harmful algal blooms.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Algas/análise , DNA de Protozoário/análise , Dinoflagelados/isolamento & purificação , Técnicas Eletroquímicas/instrumentação , Técnicas Biossensoriais/instrumentação , Proliferação Nociva de Algas
10.
Harmful Algae ; 84: 27-35, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31128810

RESUMO

Ostreopsis is a toxic benthic dinoflagellate largely distributed worldwide in tropical and temperate areas. In the Mediterranean Sea, periodic summer blooms have been reported and have become a serious concern due to their direct impact on human health and the environment. Current microalgae identification is performed via light microscopy, which is time-consuming and is not able to differentiate among Ostreopsis species. Therefore, there is mature need for rapid, specific and easy-to-use detection tools. In this work, a colorimetric assay exploiting a combination of recombinase polymerase amplification (RPA) and a sandwich hybridisation assay was developed for O. cf. ovata and O. cf. siamensis detection and quantification. The specificity of the system was demonstrated by cross-reactivity experiments and calibration curves were successfully constructed using genomic DNA, achieving limits of detection of 10 and 14 pg/µL for O. cf. ovata and O. cf. siamensis, respectively. The assay was applied to the analysis of planktonic and benthic environmental samples from different sites of the Catalan coast. Species-specific DNA quantifications were in agreement with qPCR analysis, demonstrating the reliability of the colorimetric approach. Significant correlations were also obtained between DNA quantifications and light microscopy counts. The approach may be a valuable tool to provide timely warnings, facilitate monitoring activities or study population dynamics, and paves the way towards the development of in situ tools for the monitoring of harmful algal blooms.


Assuntos
Colorimetria , Dinoflagelados , DNA , Humanos , Mar Mediterrâneo , Reprodutibilidade dos Testes
11.
Anal Chem ; 91(11): 7104-7111, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31042376

RESUMO

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays ( KD of 3-34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

12.
Biosens Bioelectron ; 134: 76-82, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30954929

RESUMO

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade-1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.


Assuntos
Técnicas Biossensoriais/instrumentação , DNA/análise , Nucleotídeos de Desoxiadenina/química , Técnicas Eletroquímicas/instrumentação , Compostos Ferrosos/química , Metalocenos/química , Reação em Cadeia da Polimerase/instrumentação , Desenho de Equipamento , Limite de Detecção , Microeletrodos , Oxirredução , Água do Mar/análise
13.
Food Chem ; 287: 354-362, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30857710

RESUMO

In this work, a duplex PCR-Enzyme Linked Oligonucleotide Assay (ELONA) is reported for the sensitive and reliable detection of pork adulteration in beef and chicken products, two of the most widely consumed meat types in the world. The strategy relies on the use of species-specific tailed primers for duplex amplification and simple dilution of the PCR reactions for direct colorimetric detection via hybridization, eliminating the need for any other post-amplification steps. A high sensitivity was achieved, with as low as 71-188 pg of genomic DNA able to be detected using mixtures of control DNA from each species. The strategy was validated using DNA add-mixtures as well as DNA extracted from raw meat mixtures and 0.5-1% w/w pork could be easily detected when mixed with beef or chicken. The proposed approach is simple, sensitive and cost-effective compared to equivalent commercial kits suitable for detecting adulterant pork levels in meat products.


Assuntos
Contaminação de Alimentos/análise , Reação em Cadeia da Polimerase/métodos , Carne Vermelha/análise , Suínos/genética , Animais , Bovinos , Galinhas , Primers do DNA , Análise de Alimentos/métodos , Oligonucleotídeos , Produtos Avícolas/análise , Sensibilidade e Especificidade , Especificidade da Espécie
14.
Anal Chem ; 90(21): 12745-12751, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30296053

RESUMO

High-risk pathogens such as Francisella tularensis and Yersinia pestis are categorized as highly hazardous organisms that can be used as biological weapons. Given the extreme infectivity of these potential biowarfare agents, a rapid, sensitive, cost-effective, and specific method for their detection is required. Here, we report the multiplexed amplification detection of genomic DNA from Francisella tularensis and Yersinia pestis. Amplification was achieved using isothermal recombinase polymerase amplification, exploiting tailed primers, followed by detection using a nucleic-acid lateral flow assay. Excess primers were removed using a novel fishing strategy, avoiding the use of postamplification purification that requires centrifugation and infers additional assay cost. The entire assay is completed in less than 1 h, achieving limits of detection of 243 fg (1.21 × 102 genome equivalent) and 4 fg (0.85 genome equivalent) for Francisella tularensis and Yersinia pestis, respectively.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Bioensaio/métodos , DNA/genética , Francisella tularensis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Yersinia pestis/isolamento & purificação , DNA/isolamento & purificação , Proteínas de Ligação a DNA/química , Endopeptidase K/química , Francisella tularensis/genética , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Proteólise , Yersinia pestis/genética
15.
Anal Chim Acta ; 1039: 140-148, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30322545

RESUMO

Karlodinium is a dinoflagellate responsible for fish-killing events worldwide. In Alfacs Bay (NW Mediterranean Sea), the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. This work presents a method that combines recombinase polymerase amplification (RPA) with an enzyme-linked oligonucleotide assay (ELONA) to identify, discriminate and quantify these two species. The system was characterised using synthetic DNA and genomic DNA, and the specificity was confirmed by cross-reactivity experiments. Calibration curves were constructed using 10-fold dilutions of cultured cells, attaining a limit of detection of around 50,000 cells/L, far below the Karlodinium spp. alert threshold (200,000 cells/L). Finally, the assay was applied to spiked seawater samples, showing an excellent correlation with the spiking levels and light microscopy counts. This approach is more rapid, specific and user-friendly than traditional microscopy techniques, and shows great promise for the surveillance and management of harmful algal blooms.


Assuntos
Ensaio de Imunoadsorção Enzimática , Toxinas Marinhas/análise , Microalgas/química , Oligonucleotídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Oligonucleotídeos/metabolismo
17.
ACS Infect Dis ; 4(9): 1306-1315, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-29972299

RESUMO

Trichomoniasis, caused by Trichomonas vaginalis, is the leading nonviral sexually transmitted infection worldwide. We report the selection of a DNA aptamer against a T. vaginalis adhesion protein, AP65, using a microtiter plate-based in vitro combinatorial chemistry process termed systematic evolution of ligands by exponential enrichment. The enriched library pool was sequenced by next-generation sequencing, and several aptamer candidates with high affinity and specificity were identified. The aptamer with the highest affinity and specificity had a KD in the low nanomolar range, as confirmed by three different techniques: surface plasmon resonance, enzyme-linked aptamer assay, and biolayer interferometry. The selected aptamer was demonstrated to have a high specificity to the AP65 protein and to T. vaginalis cells with no cross-reactivity to other enteric and urogenital microorganisms. Current work is focused on the development of inexpensive and easy-to-use aptamer-based diagnostic assays for the reliable and rapid detection of T. vaginalis in vaginal swabs.


Assuntos
Moléculas de Adesão Celular/análise , Proteínas de Protozoários/análise , Técnica de Seleção de Aptâmeros/métodos , Vaginite por Trichomonas/diagnóstico , Trichomonas vaginalis/isolamento & purificação , Moléculas de Adesão Celular/genética , Feminino , Humanos , Proteínas de Protozoários/genética , Profissionais do Sexo , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/genética
18.
Anal Biochem ; 556: 16-22, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29920236

RESUMO

DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2 min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70 fM for a ssDNA template was achieved using 45 min amplification, whilst for a dsDNA template, just 30 min amplification resulted in a slightly lower detection limit of 14 fM, whilst both 20 and 45 min amplification times were observed to provide detection limits of 71 and 72 fM, respectively, but 30 and 45 min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.


Assuntos
Doença Celíaca/genética , Primers do DNA/genética , Técnicas Eletroquímicas/métodos , Cadeias beta de HLA-DQ/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Alelos , Humanos
19.
Biosens Bioelectron ; 117: 201-206, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29906767

RESUMO

Polyoxymetalates (POMs) ([SiW11O39{Sn(CH2)2CO)}]4- and [P2W17O61{Sn(CH2)2CO)}]6-) were used to modify dideoxynucleotides (ddNTPs) through amide bond formation, and applied to the multiplexed detection of single nucleotide polymorphisms (SNPs) in an electrochemical primer extension reaction. Each gold electrode of an array was functionalised with a short single stranded thiolated DNA probe, specifically designed to extend with the POM-ddNTP at the SNP site to be interrogated. The system was applied to the simultaneous detection of 4 SNPs within a single stranded 103-mer model target generated using asymmetric PCR, highlighting the potential of POM-ddNTPs for targeted, multiplexed SNP detection. The four DNA bases were successfully labelled with both ([SiW11O39{Sn(CH2)2CO)}]4- and [P2W17O61{Sn(CH2)2CO)}]6-), and [SiW11O39{Sn(CH2)2CO)}]4- demonstrated to be the more suitable due to its single oxidation peak, which provides an unequivocal signal. The POM-ddNTP enzymatically incorporated to the DNA anchored to the surface was visualised by AFM using gold coated mica. The developed assay has been demonstrated to be highly reproducible, simple to carry out and with very low non-specific background signals. Future work will focus on applying the developed platform to the detection of SNPs associated with rifampicin resistance in real samples from patients suffering from tuberculosis.


Assuntos
Técnicas Biossensoriais/métodos , Polimorfismo de Nucleotídeo Único , Compostos de Tungstênio/química , Primers do DNA/química , Humanos , Reprodutibilidade dos Testes
20.
Chemistry ; 24(43): 11177-11184, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29782690

RESUMO

Self-assembled monolayers formed by chemisorption of thiolated molecules on gold surfaces are widely applied for biosensing. Moreover, and due to the low stability of thiol-gold chemistry, contributions to the functionalisation of gold substrates with linkers that provide a more stable platform for the immobilisation of electroactive or biological molecules are highly appreciated. Herein, it is demonstrated that a carboxylated organotin compound can be successfully grafted onto gold substrates to form a highly stable organic layer with reactivity for subsequent binding to an aminated molecule. A battery of techniques were used to characterise the surface chemistry. The grafted layer was used to anchor aminoferrocene and subjected to both thermostability tests and long-term stability studies over a period of one year, demonstrating thermostability up to 90 °C and storage stability for at least 12 months at 4 °C protected from light. The stable surface tethering of molecules on gold substrates can be exploited in a plethora of applications, including molecular techniques, such as solid-phase amplification and solid-phase melting curve analysis, that require elevated temperature stability, as well as biosensors, which require long-term storage stability.

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