Heat-transfer-based detection of L-nicotine, histamine, and serotonin using molecularly imprinted polymers as biomimetic receptors.

In this work, we will present a novel approach for the detection of small molecules with molecularly imprinted polymer (MIP)-type receptors. This heat-transfer method (HTM) is based on the change in heat-transfer resistance imposed upon binding of target molecules to the MIP nanocavities. Simultaneously with that technique, the impedance is measured to validate the results. For proof-of-principle purposes, aluminum electrodes are functionalized with MIP particles, and L-nicotine measurements are performed in phosphate-buffered saline solutions. To determine if this could be extended to other templates, histamine and serotonin samples in buffer solutions are also studied. The developed sensor platform is proven to be specific for a variety of target molecules, which is in agreement with impedance spectroscopy reference tests. In addition, detection limits in the nanomolar range could be achieved, which is well within the physiologically relevant concentration regime. These limits are comparable to impedance spectroscopy, which is considered one of the state-of-the-art techniques for the analysis of small molecules with MIPs. As a first demonstration of the applicability in biological samples, measurements are performed on saliva samples spiked with L-nicotine. In summary, the combination of MIPs with HTM as a novel readout technique enables fast and low-cost measurements in buffer solutions with the possibility of extending to biological samples.

A MIP-based impedimetric sensor for the detection of low-MW molecules.

Mimicking the selectivity and sensitivity of biological systems for sensor devices is of increasing interest in biomedical, environmental and chemical analysis. Synthetic materials with imprinted nanocavities, acting as highly selective artificial receptors, are a tailor-made solution in obtaining such a sensor. Incorporation of such molecularly imprinted polymers (MIPs) in a platform suitable for electrochemical measurements, can offer high sensitivity together with device miniaturization and an electronic read-out. As a proof of principle, a MIP-based sensor for L-nicotine has been developed. To this end, the molecular structure of L-nicotine was imprinted in a polymer matrix of polymethacrylic acid (PMAA). Subsequently, microparticles of the imprinted polymer were immobilized on thin films of the conjugated polymer OC(1)C(10)-PPV. These films were incorporated in an impedimetric sensing device. Using electrochemical impedance spectroscopy, the real part of the impedance was monitored for various concentrations. This setup allows for the detection of l-nicotine from 1 to 10 nM and is insensitive for the resembling molecule L-cotinine.

The del(2)(q32.2q33) deletion syndrome defined by clinical and molecular characterization of four patients.

We report four patients with an interstitial deletion of chromosome 2q32-->2q33. They presented similar clinical findings including pre- and postnatal growth retardation, distinct facial dysmorphism, thin and sparse hair and fair built, micrognathia, cleft or high palate, relative macroglossia, dacrocystitis, persisting feeding difficulties, inguinal hernia and broad based gait. All were severely mentally retarded. Three patients had a specific behavioral phenotype with hyperactivity and motor restlessness, chaotic behavior, happy-personality but with periods of aggression and anxiety, sleeping problems and self-mutilation. (head-banging). Array CGH and fluorescence in situ hybridization (FISH) allowed us to delineate the deletion size and showed that the four patients share a 8.1 Mb minimal deleted region. Reviewing additional nine case reports of patients with similar deletions showed striking phenotypic similarities which enabled the delineation of the 2q32.2q33 syndrome. Deletion of 2q32 has been also associated with the wrinkly skin syndrome (WWS) and isolated cleft palate. Although the patients presented here shared many aspects of WWS, they did not had the wrinkly skin. All patients had a cleft or high palate, most likely as a result of hemizygosity for SATB2. A potential commonly deleted interval of the three patients with behavioral problems, excluding the deletion in the patient without behavioral problems, is at most 0.5 Mb in size harboring only two genes.

Impedimetric immunosensors based on the conjugated polymer PPV.

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.

Partial trisomy 11q syndrome (11q23.1-->11qter) due to de novo t (11q; 13q) detected by multicolor fluorescence in situ hybridisation.

In this report we describe the identification of a de novo 46, XX, 13q + by multicolour fluorescence in situ hybridisation (M-FISH), as a partial distal 11q trisomy (11q23.1-->11qter). The clinical phenotype association with this distal 11q trisomy is briefly reviewed.

Physical map of a 1.5 mb region on 12p11.2 harbouring a synpolydactyly associated chromosomal breakpoint.

Synpolydactyly (SPD) is a rare malformation of the distal limbs known to be caused by mutations in HOXD13. We have previously described a complex form of SPD associated with synostoses in three members of a Belgian family, which co-segregates with a t(12;22)(p11.2;q13.3) chromosomal translocation. The chromosome 12 breakpoint of this translocation maps to 12p11.2 between markers D12S1034 and D12S1596. Here we show that a mutation in the HOXD13 gene is not responsible for the phenotype, and present a physical map of the region around the 12p11.2 breakpoint. Starting from D12S1034 and D12S1596, we have established a contig approximately 1.5 Mb in length, containing 13 YAC clones, 16 BAC clones, and 11 cosmid clones. FISH analysis shows that cosmid LL12NCO1-149H4 maps across the breakpoint, and Southern blot experiments using fragments of this cosmid as probes identify a rearranged BamHI fragment in the patients carrying the translocation. A search for expressed sequences within the contig have so far revealed one CpG island, seven anonymous ESTs and three previously characterised genes, DAD-R, KRAG and HT21, all of which were found not to be directly disrupted by the translocation. The gene represented by EST R72964 was found to be disrupted by the translocation. These findings lay the groundwork for further efforts to characterise a gene critical for normal distal limb development that is perturbed by this translocation.

Rapid assessment of the stability of DNA duplexes by impedimetric real-time monitoring of chemically induced denaturation.

In this article, we report on the electronic monitoring of DNA denaturation by NaOH using electrochemical impedance spectroscopy in combination with fluorescence imaging as a reference technique. The probe DNA consisting of a 36-mer fragment was covalently immobilized on nanocrystalline-diamond electrodes and hybridized with different types of 29-mer target DNA (complementary, single-nucleotide defects at two different positions, and a non-complementary random sequence). The mathematical separation of the impedimetric signals into the time constant for NaOH exposure and the intrinsic denaturation-time constants gives clear evidence that the denaturation times reflect the intrinsic stability of the DNA duplexes. The intrinsic time constants correlate with calculated DNA-melting temperatures. The impedimetric method requires minimal instrumentation, is label-free and fast with a typical time scale of minutes and is highly reproducible. The sensor electrodes can be used repetitively. These elements suggest that the monitoring of chemically induced denaturation at room temperature is an interesting approach to measure DNA duplex stability as an alternative to thermal denaturation at elevated temperatures, used in DNA-melting experiments and single nucleotide polymorphism (SNP) analysis.

A familial complex chromosome translocation resulting in duplication of 6p25.

We report on a girl with psychomotor retardation, severe speech developmental delay and mild dysmorphic features. Molecular cytogenetic analysis showed that the patient was carrier of an insertion (6)(p22.5-->22.4) in chromosome 12. Analysis of the chromosomes of the mother revealed the presence of a complex chromosomal rearrangement. In addition to the insertion (6)(p22.5-->22.4) in chromosome 12 and a pericentric inversion in chromosome 12, the 6p subtelomeric region was absent in the mother. This is, to our knowledge, the smallest pure duplication of chromosome 6p as well as the smallest cryptic subtelomeric 6pter deletion thus far reported.

Physical mapping of the t(12;22) translocation breakpoints in a family with a complex type of 3/3'/4 synpolydactyly.

We previously reported clinical and radiological findings in a Belgian family with a complex type of synpolydactyly associated with metacarpal and metatarsal synostoses, cosegregating with a balanced t(12;22). Recently, expansions of a polyalanine stretch within the first exon of the HOXD13 gene, which resides on chromosome 2q31, have been shown to cause synpolydactyly (SPD). Using exon amplification followed by direct sequencing, we were able to exclude the direct involvement of the HOXD13 gene in this family. As a first step toward the positional cloning of a candidate disease gene on chromosome 12 and/or 22 responsible for the type of complex synpolydactyly observed in this family, we report here the construction of a somatic cell hybrid retaining only the der(22) of the t(12;22)(p11.3;q13.3). STS content mapping and FISH experiments allowed us to position the chromosomal breakpoints between markers D12S1596 and D12S1034 on chromosome 12 and markers N73F4 and D22S158 on chromosome 22.

Isolation of cosmids corresponding to the chromosome breakpoints of a de novo autosomal translocation, t(6;19)(p21;q13.1), in a patient with multicystic renal dysplasia.

Hydronephrosis caused by pelvi-ureteric junction obstruction (PUJO) is a frequent urological malformation assumed to result from a deficient development of the ureteric bud. The exact etiology of pelvi-ureteric junction stenosis is unknown, but there is convincing evidence for a genetic cause, with linkage analysis predicting a hereditary hydronephrosis locus on chromosome 6p. We encountered a patient with a de novo autosomal t(6;19)(p21;q13.1) and attendant bilateral multicystic renal dysplasia (MRD), bilateral PUJO resulting in massive hydronephrosis, and an associated von Mayer-Rokitansky-Kuster disorder. On the basis of the presumption that in this patient the putative hydronephrosis gene might be disrupted by the translocation, we sought to isolate DNA from the breakpoint regions as the initial step in a strategy to identify genes affected by the t(6; 19). Using sequential rounds of fluorescence in situ hybridization (FISH) with cosmids selected from a detailed integrated map of the long arm of chromosome 19, we have identified a cosmid clone that spans the breakpoint. The position of the breakpoint was further localized by Southern blot analysis. Using a vectorette PCR approach, rearranged DNA fragments were isolated and, by comparative nucleotide sequence analysis, these were shown to contain ectopic sequences. A cosmid clone containing these ectopic sequences was isolated and shown by CASH (chromosome assignment using somatic cell hybrids) and FISH (fluorescence in situ hybridization) analysis to map to the short arm of chromosome 6 and to span the breakpoint found in the MRD patient. The isolated cosmid clones are useful reagents for analysis of other MRD patients and for the search for genes at or flanking the breakpoints.