Analytical methods

Scientific publications - Analytical methods

Reichwald, K., Hatzack, F.

"Application of a modified Haug and Lantzsch method for the rapid and accurate photometrical phytate determination in soybean, wheat, and maize meals"

Journal of Agricultural and Food Chemistry, 56 (9), pp. 2888-2891. (2008)

A modified version of the Haug and Lantzsch method for rapid photometrical phytate determination was applied for the analysis of phytate in soybean, wheat, and maize meals. In comparison to the original protocol, the amount of the toxic reagent thioglycolic acid is reduced substantially to minimize potential health risks for laboratory personnel. Different extraction conditions for soybean meal were tested, and boiling for at least 30 min was found to be necessary to remove an interfering compound in soybean meal extracts. Phytate contents determined according to the modified Haug and Lantzsch method did not differ from those obtained by measuring total precipitated phosphorus or by sensitive and specific high-performance ion chromatography. Applicability and accuracy of the modified method for phytate analysis in major feed substrates, including soy-based textured vegetable protein, were demonstrated. © 2008 American Chemical Society.

Olesen, K.-M., Hansen, S.H., Sidenius, U., Schmiegelow, K.

"Determination of leukocyte DNA 6-thioguanine nucleotide levels by high-performance liquid chromatography with fluorescence detection"

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 864 (1-2), pp. 149-155. (2008)

A HPLC method for determination of 6-thioguanine nucleotide in DNA was developed. Leukocyte DNA was isolated from peripheral blood, derivatized with chloroacetaldehyde and the formed etheno derivatives N2,3-etheno 6-thioguanine (ε6TG), 1,N6-etheno adenine (εA) and N2,3-etheno guanine (εG) were released from the DNA backbone by hydrolysis at pH 6.0 and 80 °C for 60 min. After extraction of ε6TG by immobilized metal ion affinity chromatography (IMAC) the sample was analysed by ion-pair reversed-phase HPLC with fluorescence detection. The limit of quantification was 9.0 nM and the intra- and interday precision ranged from 2.8 to 15.5%. In a small cohort of eight children with acute lymphoblastic leukaemia (ALL), a median of one 6-thioguanine base was found for each 3000 normal bases (range 1:2000-1:11000). © 2008 Elsevier B.V. All rights reserved.

Gizzi, G., Thyregod, P., Von Holst, C., Bertin, G., Vogel, K., Faurschou-Isaksen, M., Betz, R., Murphy, R., Andersen, B.B.

"Determination of phytase activity in feed: interlaboratory study"

Journal of AOAC International, 91 (2), pp. 259-267. (2008)

An interlaboratory study was conducted to determine the performance characteristics of a new method for the determination of phytase activity in feed samples. The method is based on the principle that inorganic phosphate is released from the substrate phytate under defined assay conditions and has been validated for its suitability to measure the enzyme activity of various phytase products. Two different experimental designs of the study were applied, allowing for the estimation of the precision of the method under repeatability, intermediate precision and reproducibility conditions, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.2 to 10.6% and the RSD for reproducibility (RSDR) ranged from 5.4 to 15%. The suitability of the validated method for the intended purpose was demonstrated. The obtained performance profile of the method validated in this study was comparable to that of similar methods that were exclusively validated for one phytase product.

Zarnkow, M., Mauch, A., Back, W., Arendt, E.K., Kreisz, S.

"Proso millet (Panicum miliaceum L.): An evaluation of the microstructural changes in the endosperm during the malting process by using scanning-electron and confocal laser microscopy"

Journal of the Institute of Brewing, 113 (4), pp. 355-364. (2007)

Scanning electron microscopy (SEM) and confocal scanning laser microscopy (CLSM) were used to investigate the microstructural changes in the endosperm during the malting process. SEM was a suitable tool to characterise the microstructural constitution of starch granules in proso millet and the changes occurring during malting. An early visible degradation (after 24 h) of starch granules located in the floury endosperm, which is close to the embryo, could be observed. Due to this degradation, using confocal scanning laser microscopy, a less dense packaging of this part of the endosperm was observed. Degradation of starch granules in the vitreous endosperm was obvious at a distinct later stage of malting (78 h) and a preferred attack of small granules (<2.5 μm) was detected. Changes in protein structure could not be detected by either SEM or CLSM. © 2007 The Institute of Brewing & Distilling.

Poulsen, L.K., Pedersen, M.H., Dufva, M.

"Allergology on a chip"

Clinical and Experimental Allergy, 37 (12), pp. 1736-1737. (2007)


Kim, K.-H., Brown, K.M., Harris, P.V., Langston, J.A., Cherry, J.R.

"A proteomics strategy to discover β-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry"

Journal of Proteome Research, 6 (12), pp. 4749-4757. (2007)

Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. β-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few β-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover β-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and β-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as β-glucosidases was confirmed by tandem mass spectrometry. Two novel β-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel β-glucosidase (EAL88289) was cloned and heterologously expressed. The expressed β-glucosidase showed far superior heat stability to the previously characterized β-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of β-glucosidases in a high throughput fashion. © 2007 American Chemical Society.

Råvik, M., Cimander, C., Elofsson, U., Veide, A.

"A method for microbial cell surface fingerprinting based on surface plasmon resonance"

Journal of Biochemical and Biophysical Methods, 70 (4), pp. 595-604. (2007)

A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA). © 2007 Elsevier B.V. All rights reserved.

Haack, M.B., Lantz, A.E., Mortensen, P.P., Olsson, L.

"Chemometric analysis of in-line multi-wavelength fluorescence measurements obtained during cultivations with a lipase producing Aspergillus oryzae strain"

Biotechnology and Bioengineering, 96 (5), pp. 904-913. (2007)

The filamentous fungus, Aspergillus oryzae, was cultivated in batch and fed-batch cultivations in order to investigate the use of multi-wavelength fluorescence for monitoring course of events during filamentous fungi cultivations. The A. oryzae strain applied expressed a fungal lipase from Thermomyces lanuginosus. Spectra of multi-wavelength fluorescence were collected every 5 min with the Bio View® system (DELTA, Denmark) and both explorative and predictive models, correlating the fluorescence data with cell mass and lipase activity, were built. During the cultivations, A. oryzae displayed dispersed hyphal growth and under these conditions no fouling of the multi-wavelength fluorescence sensor was observed. The scores of a parallel factor analysis (PARAFAC) model, based on the fluorescence spectra, gave clear evidence of, for example, the on-set of the feeding phase. The predictive models, estimating the cell mass, showed correlations between 0.73 and 0.97 with root mean square error of cross validation (RMSECV) values between 1.48 and 0.77 g·kg-1. A model estimating the lipase activity was also constructed for the fed-batch cultivations with a correlation of 0.93. The results presented here clearly show that multi-wavelength fluorescence is a useful tool for monitoring fed-batch cultivations of filamentous fungi. © 2006 Wiley Periodicals, Inc.

Sonesson, A.W., Callisen, T.H., Brismar, H., Elofsson, U.M.

"A comparison between dual polarization interferometry (DPI) and surface plasmon resonance (SPR) for protein adsorption studies"

Colloids and Surfaces B: Biointerfaces, 54 (2), pp. 236-240. (2007)

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30-1.35 mg/m2 at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest. © 2006 Elsevier B.V. All rights reserved.

Houmøller, L.P., Kristensen, D., Rosager, H.

"Determination of SFC, FFA, and equivalent reaction time for enzymatically interestified oils using NIRS"

Talanta, 71 (2), pp. 868-873. (2007)

The use of near infrared spectroscopy (NIRS) for rapid determination of the degree of interesterification of blends of palm stearin, coconut oil, and rapeseed oil obtained using an immobilized Thermomyces lanuginosa lipase at 70 °C was investigated. Interesterification was carried out by applying both fixed bed and batch reactors. Calibrations were developed for quantitative determination of solid fat content (SFC) at 10, 20, 30, 35, and 40 °C and free fatty acid (FFA) resulting in root mean square errors of prediction of 1.0, 1.3, 1.4, 1.6, 1.7, and 0.19% (w/w), respectively. The data showed that NIRS could be used to replace the traditional methods for determining FFA and SFC in vegetable oils. It was possible to monitor the activity of the immobilized enzyme for interesterification of margarine oils by predicting the equivalent reaction time in a batch reactor from NIR spectra. Root mean square errors of prediction for two different oil blends interesterified for 300 and 170 min were 21 and 12 min, respectively. © 2006 Elsevier B.V. All rights reserved.

Brinch-Pedersen, H., Hatzack, F.

"Analysis of phosphorus and phosphorylated compounds in the context of plant physiology and global phosphorus management: A review"

Current Analytical Chemistry, 2 (4), pp. 421-430. (2006)

The management of phosphorus (P) as a non-renewable resource is receiving increasing attention at the dawn of the new millennium. The importance of phosphate as a nutrient, the environment phosphate load in areas with intens livestock production and the depletion of the high quality phosphate resources are accentuating the need of a holistic and global phosphorus-resource management and understanding of the physiological roles, the biosynthesis and the breakdown of phosphorylated compounds in plants. Two important routes towards improved P-resource management are genetic engineering of plants towards improved P bioavailability and the application of microbial phytases to increase the bioavailability of phytate-P in monogastric animals. The present review gives an overview on these P-management approaches and discusses the arsenal of analytical techniques that can be used to analyze P and P-compounds. This analytical capability is an important determinant in the development of more advanced and more effective approaches towards P-resource management. © 2006 Bentham Science Publishers Ltd.

Mejlhede, N., Kyjovska, Z., Backes, G., Burhenne, K., Rasmussen, S.K., Jahoor, A.

"EcoTILLING for the identification of allelic variation in the powdery mildew resistance genes mlo and Mla of barley"

Plant Breeding, 125 (5), pp. 461-467. (2006)

In this investigation the successful implementation of a CEL I-based mutation detection technique for the discovery and detection of DNA polymorphism in the genes mlo and Mla of barley is described. The technique is called EcoTILLING, which is a high-throughput method to detect and discover new point mutations and small insertions/deletions in DNA. That the method not only reveals polymorphism between different alleles but can also be used as a powerful genetic marker is demonstrated. The genes mlo and Mla are involved in the defence of barley against the fungal pathogen powdery mildew. The powdery mildew resistance gene mlo is a single copy gene, whereas multiple alleles exist at the Mla locus. With EcoTILLING it was possible to identify point mutations and deletions in each of the 11 mlo mutants tested. For Mla 25 natural barley variants were tested, and although the identification was complex due to the presence of highly similar paralogues of Mla, most of the recently identified alleles from Hordeum vulgare ssp. spontaneum were identified. This method offers the possibility to combine different mlo alleles with different Mla alleles from wild barley to obtain cultivars with more durable resistance. © 2006 The Authors.

Jürgen, B., Barken, K.B., Tobisch, S., Pioch, D., Wümpelmann, M., Hecker, M., Schweder, T.

"Application of an electric DNA-chip for the expression analysis of bioprocess-relevant marker genes of Bacillus subtilis"

Biotechnology and Bioengineering, 92 (3), pp. 299-307. (2005)

The knowledge of critical process-relevant genes can be used for an improved control of bioprocesses. So far bioprocess-relevant marker genes can be analyzed by established expression analysis methods only off-line. In this study, an alternative approach for a potential at-line monitoring of gene expression during bioprocesses is suggested. This approach is based on the measurement of specific mRNAs on an electric DNA-chip in connection with a magnetic bead-based sandwich hybridization. In order to allow an at-line measurement of specific mRNAs an improved method for a fast and partially automated isolation of high quality-RNA samples was developed. The expression analysis of the electric DNA-chip was compared with optical DNA micro arrays and the real time RT-PCR for three selected process-relevant genes of Bacillus subtilis. We demonstrate that the mRNA analysis by means of the electric DNA-chip gives similar results compared to the micro array analysis and the real time RT-PCR technique. © 2005 Wiley Periodicals, Inc.

Ignatjev, I., Valincius, G., Švedaite, I., Gaidamauskas, E., Kažemekaite, M., Razumas, V., Svendsen, A.

"Direct amperometric determination of lipase activity"

Analytical Biochemistry, 344 (2), pp. 275-277. (2005)


Valincius, G., Ignatjev, I., Niaura, G., Kažemékaité, M., Talaikyté, Z., Razumas, V., Svendsen, A.

"Electrochemical method for the detection of lipase activity"

Analytical Chemistry, 77 (8), pp. 2632-2636. (2005)

A novel electrochemical technique for the general assay of lipase activity is described. The method utilizes a solid-supported lipase substrate, which is formed by dripping and drying a small amount of an ethanol solution of 9-(5′-ferrocenylpentanoyloxy)nonyl disulfide (FPONDS) onto gold modified by a hexanethiol self-assembled monolayer. The redox ferrocene group of FPONDS generates the electrochemical signal, the intensity of which is proportional to the number of FPONDS molecules at the interface. Electrochemical and surface-enhanced infrared absorption spectroscopic data, as well as control experiments with an engineered, deactivated mutant enzyme, demonstrate that the wild-type lipase from Thermomyces lanuginosus is capable of cleaving the ester bonds of FPONDS molecules via an enzymatic hydrolysis mechanism, which includes the adsorption of the lipase onto the substrate surface. The hydrolysis liberates the ferrocene groups from the interface triggering a decay of the electrochemical redox signal. The rate of the electrochemical signal decrease is proportional to the lipase activity/concentration. These data suggest a general method for the direct measure of enzymatic activity of lipases. © 2005 American Chemical Society.

Mortensen, P.P., Esbensen, K.H.

"Optimization of the Angle Measure Technique for image analytical sampling of particulate matter"

Chemometrics and Intelligent Laboratory Systems, 75 (2), pp. 219-229. (2005)

Optimization of an industrial image analytical application which uses the Angle Measure Technique (AMT) as an integrated part of estimation of the quality parameter dust level for encapsulated enzyme granules has been carried out. The principal AMT results from this study can be generalized to all similar types of segregating particulate matter. The first part of the study addresses pixel-sampling strategies within one image prior to application of the Angle Measure Technique, where the unfolded image is regarded as a one-dimensional object. Optimal parameters for the AMT algorithm is found to be systematic or stratified sampling of 500+ initiating points, unfolded in a novel spiral- or snake-wise fashion from the fluorescent image; this new unfolding procedure eliminates certain algorithmic "wrap around" artifacts which have not been sufficiently in need of rectification until now. The second part is directed towards delineating optimal image analytical sampling of a series of such images, i.e., optimization of image process sampling. The minimum number of images needed to uphold a sufficiently good online estimate of the QC parameter in question is found to be corresponding to a relatively high sampling rate of 15-20%, related to an intrinsic measurement error level of a mandated analytical reference method. Increasing the number of image increments included in the image analytical sample primarily affects the accuracy of the estimates, whereas precision is much less affected, attesting to a satisfactory stability of the image analytical method developed, which is now under industrial implementation. © 2004 Elsevier B.V. All rights reserved.

Stocks, S.M.

"Mechanism and use of the commercially available viability stain, BacLight"

Cytometry Part A, 61 (2), pp. 189-195. (2004)

Background: BacLight (Molecular Probes, Eugene, OR, USA) is a popular fluorescence-based two-component stain for determining bacterial cell viability. The main purpose of this work was to fully elucidate the mechanism and to determine why it is sometimes reported that cells stain simultaneously live and dead. Methods: Solutions of DNA were stained with the two components, propidium iodide (PI) and SYTO9, in different combinations, and fluorescence spectra were collected. Results: KPl and KSYTO9 were approximately 3.7 x 105/M and 1.8 x 105/M. SYTO9 emissions were stronger and overlapped those of PI. Fluorescence resonance energy transfer from SYTO9 to PI was observed. It was, even under normal conditions, possible for DNA bound SYTO9 to have a component in the red region equal to that of DNA bound PI. Potentially confusing emissions were also found to occur when PI was not in sufficient excess to saturate nucleic acid (>0.4 M PI to 1 M DNA base pairs). Conclusions: The mechanism is a combination of displacement of SYTO9 by PI and quenching of SYTO9 emissions by fluorescence resonance energy transfer. Confusing results can occur if the relative intensities of the stains or the concentration of PI relative to nucleic acid are not properly accounted for. © 2004 Wiley-Liss, Inc.

Hansen, E., Mollerup, J.M.

"The influence of retention on the plate height in ion-exchange chromatography"

Separation Science and Technology, 39 (9), pp. 2011-2030. (2004)

The plate heights for the amino acid tyrosine (anion exchange) and the polypeptide aprotinin (cation exchange) were determined on a porous media (Resource 15) and a gel filled media (HyperD 20) at salt concentrations ranging from weak to strong retention. At a constant velocity, measurements showed that the plate height increase with increasing retention, went through a maximum, and finally, decreased as the retention increased, i.e., when the salt concentration was lowered further. The band broadening of a chromatographic peak in the column was caused by the axial dispersion and mass transfer. In this article, the rate of mass transfer in the particles is described by three different rate mechanisms, pore diffusion, solid diffusion, and parallel diffusion. The van Deemter equation was used to model the data to determine the mass-transfer properties. The development of the plate height with increasing retention revealed a characteristic behavior for each rate mechanism. In the pore diffusion model, the plate height increased toward a constant value at strong retention, while the plate height in the solid diffusion model decreased, approaching a constant value at strong retention. In the parallel diffusion model, both pore and solid diffusion took place. Therefore, the parallel diffusion model coincides with the pore diffusion model at weak retention and with the solid diffusion model at strong retention, while a maximum is reached at intermediate retention, resulting in a bell-shaped curve. This behavior corresponds to the observed variation of the plate height at constant velocity. Neither the pore nor the solid diffusion model can describe the experimental data while a satisfactory fit was obtained using the parallel diffusion model.

Klenø, T.G., Andreasen, C.M., Kjeldal, H.Ø., Leonardsen, L.R., Krogh, T.N., Neilsen, P.F., Sørensen, M.V., Jensen, O.N.

"MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports"

Analytical Chemistry, 76 (13), pp. 3576-3583. (2004)

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (< 1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.

Grotkjær, T., Åkesson, M., Christensen, B., Gombert, A.K., Nielsen, J.

"Impact of Transamination Reactions and Protein Turnover on Labeling Dynamics in 13C-Labeling Experiments"

Biotechnology and Bioengineering, 86 (2), pp. 209-216. (2004)

A dynamic model describing carbon atom transitions in the central metabolism of Saccharomyces cerevisiae is used to investigate the influence of transamination reactions and protein turnover on the transient behavior of 13C-labeling chemostat experiments. The simulations performed suggest that carbon exchange due to transamination and protein turnover can significantly increase the required time needed for metabolites in the TCA cycle to reach isotopic steady state, which is in agreement with published experimental observations. On the other hand, transamination and protein turnover will speed-up the net rate of incorporation of labeled carbon into some free and protein-bound amino acids. The simulation results indicate that the pattern of labeled carbon incorporation into amino acids obtained from biomass hydrolysate shows significant deviation from the commonly assumed first-order kinetics behavior until after three residence times. These observations suggest that greater caution should be used while also pointing to new opportunities in the design and interpretation of 13C-labeling experiments. © 2004 Wiley Periodicals, Inc.

Navrátil, M., Cimander, C., Mandenius, C.-F.

"On-line Multisensor Monitoring of Yogurt and Filmjölk Fermentations on Production Scale"

Journal of Agricultural and Food Chemistry, 52 (3), pp. 415-420. (2004)

Near-infrared (NIR) spectrometry and electronic nose (EN) data were used for on-line monitoring of yogurt and filmjölk (a Swedish yogurt-like sour milk) fermentations under industrial conditions. The NIR and EN signals were selected by evaluation of principal component analysis loading vectors and further analyzed by studying the variability of the selected principal components. First principal components for the NIR and the EN signals were used for on-line generation of a process trajectory plot visualizing the actual state of fermentation. The NIR signals were also used to set up empirical partial least-squares (PLS) models for prediction of the cultures' pH and titratable acidity (expressed as Thorner degrees, oT). By using five or six PLS factors the models yielded acceptable predictions that could be further improved by increasing the number of reliable and precise calibration data. The presented results demonstrate that the fusion of the NIR and EN signals has a potential for rapid on-line monitoring and assessment of process quality of yogurt fermentation.

Gabig-Ciminska, M., Holmgren, A., Andresen, H., Bundvig Barken, K., Wümpelmann, M., Albers, J., Hintsche, R., Breitenstein, A., Neubauer, P., Los, M., Czyz, A., Wegrzyn, G., Silfversparre, G., Jürgen, B., Schweder, T., Enfors, S.-O.

"Electric chips for rapid detection and quantification of nucleic acids"

Biosensors and Bioelectronics, 19 (6), pp. 537-546. (2004)

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the biorecognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 1011 to 1010 molecules, were assayed within 25min and 4h, respectively. © 2003 Elsevier B.V. All rights reserved.

Barken, K.B., Gabig-Ciminska, M., Holmgren, A., Molin, S.

"Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization"

BioTechniques, 36 (1), pp. 124-132. (2004)

Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5′ end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-aminophenol, which is produced by alkaline phosphatase, a detection limit of 2 × 10-13 M mRNA molecules was reached without the use of a nucleic acid amplification step.