Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR

Feriotto, Giordana; Ferlini, Alessandra; Ravani, Anna; Calzolari, Elisa; Mischiati, Carlo; Bianchi, Nicoletta; Gambari, Roberto

doi:10.1002/humu.1151

Cited by 36 publications

(38 citation statements)

References 31 publications

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“…[8][9][10] However, to our knowledge, no report was published on the parallel diagnosis of multiple mutations. This is a very important feature of diagnosis protocols, since in most cases, hereditary diseases involving point mutations are highly heterogeneous.…”

Section: Discussionmentioning

confidence: 99%

“…After hybridization, the sensor chips were regenerated by performing a 5 ml pulse of 50 mM NaOH. 8,10 Sensorgrams were analyzed with the BIAevaluationt 2.1 software. 6 Blank subtractions were performed in all the experiments reported.…”

Section: Surface Plasmon Resonancementioning

confidence: 99%

“…In any case, we expect that the probes recognizing betathalassemia mutations will either be more efficient than the normal probes in hybridizing with full matched target sequences (for instance, in hybridizing with PCR products from homozygous thalassemia patients), or will generate more stable hybrids, as reported by our group for SPR-based analysis of single-point mutations. 8,10 Furthermore, the same probe (specific for either normal or mutated sequence) might behave differently if injected in flow cells carrying PCR products with different mutations and, therefore, different secondary structures.…”

Section: Experimental Strategy and Expected Secondary Structures Of Tmentioning

confidence: 99%

“…For instance, we have recently published two reports on the use of surface plasmon resonance (SPR)-based BIA and oligonucleotide (ODN) probes to detect cystic fibrosis deltaF508 and W1282X mutations [8][9][10] present in target DNA amplified by polymerase chain reaction (PCR). 11 The conclusions of these two studies were that (a) the ability of ODN probes to discriminate efficiently in SPR-based BIA between target sequences differing in terms of a single nucleotide largely depends on length; (b) short ODNs (9-10 mers) perform differential hybridization thereby being suitable for diagnostics, but, unfortunately, they could be inefficient in hybridizing with single-stranded PCR products exhibiting heavy secondary structures; (c) longer oligonucleotide probes (12-13 mers) are not informative during the hybridization step (they hybridize to both normal and mutated DNAs), but could be used, with excellent results, in the dissociation phase, since stability of mismatched hybrids is very low; and (d) 15-20 mer ODN probes do not discriminate between full matched and mismatched target DNA in BIA experiments, yielding in both cases stable hybrids.…”

mentioning

confidence: 99%

“…[8][9][10]15,[18][19][20] The aim of this study was to verify whether SPRbased BIA could be employed in multiplex analysis for real-time detection of several mutations of the human beta-globin gene causing beta-thalassemia.…”

mentioning

confidence: 99%

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Real-time multiplex analysis of four beta-thalassemia mutations employing surface plasmon resonance and biosensor technology

Feriotto

Breveglieri

Finotti

et al. 2004

Laboratory Investigation

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In this paper, biospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies was applied to the analysis of multiple mutations of the human beta-globin gene. To this aim, large target polymerase chain reaction (PCR) products were immobilized on sensor chips and then probes detecting beta139 (C4T), beta1IVSI-1 (G4A), beta þ IVSI-6 (T4C) and beta þ IVSI-110 (G4A) thalassemia mutations were sequentially injected. In this study, a total of ten normal and seven heterozygous subjects, and six homozygous patients were considered. The results obtained allow to conclude that discrimination between normal subjects, heterozygous, and homozygous patients is readily achieved for all the four mutations by PCR amplification of genomic DNA containing all the regions corresponding to the same mutations, immobilization of the same PCR products, and hybridization. To our knowledge the procedure described here is the first reported on the use of SPR-based BIA and biosensor technology for multiple detections of point mutations.

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Section: Discussionmentioning

confidence: 99%

Section: Surface Plasmon Resonancementioning

confidence: 99%

Section: Experimental Strategy and Expected Secondary Structures Of Tmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Real-time multiplex analysis of four beta-thalassemia mutations employing surface plasmon resonance and biosensor technology

Feriotto

Breveglieri

Finotti

et al. 2004

Laboratory Investigation

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PNA Molecular Beacons Assembled by Post‐Synthetic Click Chemistry Functionalization

Wang

Hudson

2015

ChemBioChem

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To avoid the tedious synthesis of functionalized peptide nucleic acid (PNA) monomers for probe development, we proposed a simple approach to modify PNA oligomers by post-synthetic on-resin click chemistry. PNA molecular beacons (MBs) were prepared by incorporation of azide-containing monomers into the oligomer by automatic solid-phase peptide synthesis and subsequent derivatization with pyrene moieties by copper-catalyzed azide-alkyne cycloaddition. Two pyrene-based quencher-free PNA molecular beacons, a stemless MB and one possessing a stem-loop structure, targeting a portion of the cystic fibrosis gene, were successfully synthesized by using this method. Fluorescence studies showed that the stem-loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct.

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Electrochemical Immunosensors for the Rapid Screening of Cystic Fibrosis and Duchenne Muscular Dystrophy

et al. 2017

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Cystic Fibrosis (CF) and Duchenne Muscular Dystrophy (DMD) are well characterized progressive inherited diseases associated with significant morbidity and mortality. Therefore, the early, rapid and affordable diagnosis of these disorders through newborn screening is highly important for the appropriate management. Here, we report label‐free impedance immunosensors for the simple screening of CF and DMD through the detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein fragment and a peptide sequence for dystrophin (DMD). The biosensors were constructed by the covalent immobilization of specific antibodies for CFTR and DMD on standard gold (Au) electrodes. The immunosensors response was measured based on the change in the electrochemical impedance spectroscopy (EIS) signals after binding with the peptides. The specific recognition of the immunosensor surfaces to the target antigens leads to retardation of the access of ferri‐ferrocyanide redox molecules to the surface and thus, enhances the charge transfer resistance (Rct). These impedimetric immunosensors enabled sensitive, fast, selective and accurate estimation of CFTR and DMD levels within a linear range from 1.0 pg/mL to 1 μg/mL and 1.0 pg/mL to 10 ng/mL with lower detection limits of 0.8 and 0.7 pg/mL for CFTR and DMD, respectively. Moreover, the immunosensors were tested for the detection of CFTR and DMD in human serum showing very good agreement with enzyme‐linked immunosorbent assays (ELISA). This work represents a novel low cost analytical method that aims to satisfy the unmet public health need in the early diagnosis of CF and DMD and can be extended to detect other hereditary disorders.

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Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR

Cited by 36 publications

References 31 publications

Real-time multiplex analysis of four beta-thalassemia mutations employing surface plasmon resonance and biosensor technology

Real-time multiplex analysis of four beta-thalassemia mutations employing surface plasmon resonance and biosensor technology

PNA Molecular Beacons Assembled by Post‐Synthetic Click Chemistry Functionalization

Electrochemical Immunosensors for the Rapid Screening of Cystic Fibrosis and Duchenne Muscular Dystrophy

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