| 1. |
- Acero Sanchez, Josep Ll., et al.
(författare)
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Electrochemical Genetic Profiling of Single Cancer Cells
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:6, s. 3378-3385
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Tidskriftsartikel (refereegranskat)abstract
- Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.
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| 2. |
- Beni, Valerio, et al.
(författare)
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Gold nanoparticle fluorescent molecular beacon for low-resolution DQ2 gene HLA typing
- 2012
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Berlin/Heidelberg. - 1618-2642 .- 1618-2650. ; 402:3, s. 1001-1009
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Tidskriftsartikel (refereegranskat)abstract
- Coeliac disease is an inflammation of the small intestine triggered by gluten ingestion. We present a fluorescent genosensor, exploiting molecular-beacon-functionalized gold nanoparticles, for the identification of human leukocyte antigen (HLA) DQ2 gene, a key genetic factor in coeliac disease. Optimization of sensor performance was achieved by tuning the composition of the oligonucleotide monolayer immobilized on the gold nanoparticle and the molecular beacon design. Co-immobilization of the molecular beacon with a spacing oligonucleotide (thiolated ten-thymine oligonucleotide) in the presence of ten-adenine oligonucleotides resulted in a significant increase of the sensor response owing to improved spacing of the molecular beacons and extension of the distance from the nanoparticle surface, which renders them more available for recognition. Further increase in the response (approximately 40%) was shown to be achievable when the recognition sequence of the molecular beacon was incorporated in the stem. Improvement of the specificity of the molecular beacons was also achieved by the incorporation within their recognition sequence of a one-base mismatch. Finally, gold nanoparticles functionalized with two molecular beacons targeting the DQA1*05* and DQB1*02* alleles allowed the low-resolution typing of the DQ2 gene at the nanomolar level.
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| 3. |
- Joda, Hamdi, et al.
(författare)
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DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection
- 2015
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 474, s. 66-68
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Tidskriftsartikel (refereegranskat)abstract
- We report on a simple approach to enhance solid-phase hybridization-based single base mismatch discrimination at high ionic strength based on the deliberate insertion of a natural DNA base mismatch in the surface-tethered probe. A large drop in hybridization signal of single base mismatched alleles using the designed probe as compared with the conventional probe, from 80% to less than 25% of the signal obtained with the fully complementary, non-mutation-containing sequence, when using colorimetric detection was further improved to 20% when using electrochemical detection, attributable to a difference of spacing of immobilized probes. Finally, the designed probe was used for the electrochemical detection of the DQA1*05:05 allele amplified from real human blood samples.
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| 4. |
- Joda, Hamdi, et al.
(författare)
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Low-medium resolution HLA-DQ2/DQ8 typing for coeliac disease predisposition analysis by colorimetric assay
- 2012
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Berlin/Heidelberg. - 1618-2642 .- 1618-2650. ; 403:3, s. 807-819
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Tidskriftsartikel (refereegranskat)abstract
- Coeliac disease is an inflammation of the small intestine, occurring in genetically susceptible individuals triggered by the ingestion of gluten. Human Leukocyte Antigens (HLA) DQ2 and DQ8 gene have been identified as key genetic factors in coeliac disease as they are presented in almost 100 % of the patients. These genes are encoded by the combination of certain alleles in the DQA and DQB region of chromosome 6. Specifically, DQA1*05:01 and DQB1*02:01 alleles for serologically defined leukocyte antigen DQ2 cis, DQA1*05:05 and DQB1*02:02 for DQ2 trans and DQA1*03:01 and DQB1*03:02 alleles for the DQ8. Specific identification of these alleles is a challenge due to the high number of alleles that have been identified so far: 46 in the DQA region and 160 in the DQB region (as of IMGT/HLA Database 10/2011 release). In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported. Finally, the performances of the developed typing platform were validated by the analysis of real patient samples and HLA typing, compared with those obtained using hospital based typing technology and an excellent correlation obtained.
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| 5. |
- Joda, Hamdi, et al.
(författare)
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Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease
- 2014
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Berlin/Heidelberg. - 1618-2642 .- 1618-2650. ; 406:12, s. 2757-2769
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Tidskriftsartikel (refereegranskat)abstract
- Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.
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