Miniaturised laboratory-on-chip systems promise rapid, sensitive, and multiplexed detection of biological samples for medical diagnostics, drug discovery, and high-throughput screening. Using micro-fabrication techniques and incorporating a unique design of transistor-based heating, researchers at the University of Illinois at Urbana-Champaign are further advancing the use of silicon transistor and electronics into chemistry and biology for point-of-care diagnostics.
Lab-on-a-chip technologies are attractive as they require fewer reagents, have lower detection limits, allow for parallel analyses, and can have a smaller footprint.
‘Integration of various laboratory functions onto microchips has been intensely studied for many years,’ explained Rashid Bashir, an Abel Bliss Professor of electrical and computer engineering and of bioengineering at Illinois. ‘Further advances of these technologies require the ability to integrate additional elements, such as the miniaturised heating element, and the ability to integrate heating elements in a massively parallel format compatible with silicon technology.
‘In this work, we demonstrated that we can heat nanoliter volume droplets, individually and in an array, using VLSI silicon based devices, up to temperatures that make it interesting to do various biochemical reactions within these droplets.’
‘Our method positions droplets on an array of individual silicon microwave heaters on chip to precisely control the temperature of droplets-in-air, allowing us to perform biochemical reactions, including DNA melting and detection of single base mismatches,’ said Eric Salm, first author of the paper.
According to Salm, approaches to perform localised heating of these individual subnanoliter droplets can allow for new applications that require parallel, time-, and space multiplex reactions on a single integrated circuit. Within miniaturised laboratory-on-chips, static and dynamic droplets of fluids in different immiscible media have been used as individual vessels to perform biochemical reactions and confine the products.
‘This technology makes it possible to do cell lysing and nucleic acid amplification reactions within these individual droplets – the droplets are the reaction vessels or cuvettes that can be individually heated,’ Salm added.
‘We also demonstrate that ssDNA probe molecules can be placed on heaters in solution, dried, and then rehydrated by ssDNA target molecules in droplets for hybridisation and detection,’ said Bashir, who is director of the Micro and Nanotechnology Laboratory at Illinois. ‘This platform enables many applications in droplets including hybridisation of low copy number DNA molecules, lysing of single cells, interrogation of ligand–receptor interactions, and rapid temperature cycling for amplification of DNA molecules.
‘Notably,’ Bashir added, ‘our miniaturised heater could also function as dual heater/sensor elements, as these silicon-on-insulator nanowire or nanoribbon structures have been used to detect DNA, proteins, pH, and pyrophosphates.
By using microfabrication techniques and incorporating the unique design of transistor-based heating with individual reaction volumes, ‘laboratory-on-a-chip’ technologies can be scaled down to ‘laboratory-on-a-transistor’ technologies as sensor/heater hybrids that could be used for point-of-care diagnostics.’ University of Illinois at Urbana-Champaign

Tiny biomolecular chambers called nanopores that can be selectively heated may help doctors diagnose disease more effectively if recent research by a team at the National Institute of Standards and Technology (NIST), Wheaton College, and Virginia Commonwealth University (VCU) proves effective. Though the findings may be years away from application in the clinic, they may one day improve doctors’ ability to search the bloodstream quickly for indicators of disease—a longstanding goal of medical research.
The team has pioneered work on the use of nanopores—tiny chambers that mimic the ion channels in the membranes of cells—for the detection and identification of a wide range of molecules, including DNA. Ion channels are the gateways by which the cell admits and expels materials like proteins, ions and nucleic acids. The typical ion channel is so small that only one molecule can fit inside at a time.
Previously, team members inserted a nanopore into an artificial cell membrane, which they placed between two electrodes. With this set-up, they could drive individual molecules into the nanopore and trap them there for a few milliseconds, enough to explore some of their physical characteristics.
‘A single molecule creates a marked change in current that flows through the pore, which allows us to measure the molecule’s mass and electrical charge with high accuracy,’ says Joseph Reiner, a physicist at VCU who previously worked at NIST. ‘This enables discrimination between different molecules at high resolution. But for real-world medical work, doctors and clinicians will need even more advanced measurement capability.’
A goal of the team’s work is to differentiate among not just several types of molecules, but among the many thousands of different proteins and other biomarkers in our bloodstream. For example, changes in protein levels can indicate the onset of disease, but with so many similar molecules in the mix, it is important not to mistake one for another. So the team expanded their measurement capability by attaching gold nanoparticles to engineered nanopores, ‘which provides another means to discriminate between various molecular species via temperature control,’ Reiner says.
The team attached gold nanoparticles to the nanopore via tethers made from complementary DNA strands. Gold’s ability to absorb light and quickly convert its energy to heat that conducts into the adjacent solution allows the team to alter the temperature of the nanopore with a laser at will, dynamically changing the way individual molecules interact with it.
‘Historically, sudden temperature changes were used to determine the rates of chemical reactions that were previously inaccessible to measurement,’ says NIST biophysicist John Kasianowicz. ‘The ability to rapidly change temperatures in volumes commensurate with the size of single molecules will permit the separation of subtly different species. This will not only aid the detection and identification of biomarkers, it will also help develop a deeper understanding of thermodynamic and kinetic processes in single molecules.’ EurekAlert

A gene linked to autism spectrum disorders that was manipulated in two lines of transgenic mice produced mature adults with irreversible deficits affecting either learning or social interaction.
The findings have implications for potential gene therapies but they also suggest that there may be narrow windows of opportunity to be effective, says principal investigator Philip Washbourne, a professor of biology and member of the University of Oregon’s Institute of Neuroscience.
The research, reported by an 11-member team from three universities, targeted the impacts of alterations in the gene neuroligin 1 — one of many genes implicated in human autism spectrum disorders — to neuronal synapses in the altered mice during postnatal development and as they entered adulthood. One group over-expressed the normal gene, the other a mutated version.
Mice with higher-than-normal levels of the normal gene after a month had skewed synapses at maturity. Many were larger, appearing more mature, than normal. In these mice, Washbourne said, there were clear cognitive problems. ‘Behaviour was just not normal. They didn’t learn very well, and they were slower to learn, but their social behaviour was not impacted.’
Mice over-producing a mutated version of the gene reached adulthood with structurally immature synapses. ‘They were held back in development and behaviour — the way they behave in terms of learning and memory, in terms of social interaction,’ he said. ‘These were adult mice, three months old, but they behaved like normal mice at four weeks old. We saw arrested development. Learning is a little bit better, they are more flexible just like young mice, they learn faster, but their social interaction is off. To us, this looked more like Asperger’s syndrome.
‘So with the same gene, doing two different manipulations — over-expressing the normal form or over-expressing a mutated form — we’ve gone to two different ends of the autism spectrum,’ said Washbourne, whose lab focuses on basic synapse formation and what goes wrong in relationship to autism. Work has been done in both mice and zebra fish.
‘We made these mice so that we can turn the genes on and off as we want,’ Washbourne said. ‘Using an antibiotic, doxycycline, it turns off these altered genes that we inserted into their chromosomes. While on doxycycline, the mice are absolutely normal.’
However, if the inserted gene was turned off after the completion of development, mice still showed altered synapses and behaviour. This result suggests that any kind of gene therapy may have to be applied to individuals with autism early on.
Effects seen in the social behaviour of mice with the mutated gene, he said, are not unlike observations reported by parents of many autistic children. While normal mice prefer to engage with new mice entering their world rather than familiar others, or even a new inanimate object, these mice split their time equally. ‘It’s not a deficit in memory regarding which mouse is which, it’s more a weighting of their interaction. Does that mean they are autistic? I don’t know, but if you talk to parents of autistic children, one of the frustrating things they report is that their children treat complete strangers in exactly the same way that they treat them.’
While the findings provide new insights, Washbourne said, any translation into treatment could be decades away. ‘A problem with autism is there are many different genes potentially involved. It could be that some day, if you are diagnosed with autism, a mouth swab might allow for the identification of the exact gene that is mutated and allow for targeted therapy,’ he said. ‘Genome sequencing already has turned up subtle mutations in lots of genes. Autism might be like cancer, with hundreds of potential combinations of faulty genes.’ University of Oregon

Johns Hopkins scientists have found out how a gout-linked genetic mutation contributes to the disease: by causing a breakdown in a cellular pump that clears an acidic waste product from the bloodstream. By comparing this protein pump to a related protein involved in cystic fibrosis, the researchers also identified a compound that partially repairs the pump in laboratory tests.
The mutation in question, known as Q141K, results from the simple exchange of one amino acid for another, but it prevents the protein ABCG2 from pumping uric acid waste out of the bloodstream and into urine. A build-up of uric acid in the blood can lead to its crystallisation in joints, especially in the foot, causing excruciatingly painful gout.
‘The protein where the mutation occurs, ABCG2, is best known for its counterproductive activity in breast cancer patients, where it pumps anti-cancer drugs out of the tumour cells we are trying to kill,’ says William Guggino, Ph.D., professor and director of the Department of Physiology at the Johns Hopkins University School of Medicine. ‘In kidney cells, though, ABCG2 is crucial for getting uric acid out of the body. What we figured out is exactly how a gout-causing genetic mutation inhibits ABCG2 function.’
Gout affects 2 to 3 percent of Americans, approximately 6 million people. It usually involves sudden attacks of severe pain, often in the joint at the base of the big toe and frequently in the wee hours of the morning, when body temperature is lowest. It has been nicknamed the ‘disease of kings,’ because it usually results from high-purine diets, food that only kings and other noblemen could afford in large quantities in bygone years: red meat, organ meats, oily fishes and some vegetables like asparagus and mushrooms.
Guggino notes that the ABCG2 Q141K mutation was first connected with gout in 2008 through a large genomic study directed, in part, by Josef Coresh, M.D., a biostatistician and epidemiologist at the Johns Hopkins University School of Public Health. At the time, Guggino’s laboratory was studying a protein frequently found mutated in cystic fibrosis patients: cystic fibrosis transmembrane conductance regulator, or CFTR. The structure of ABCG2 is quite similar to CFTR’s, so Coresh suggested that Guggino’s team apply their knowledge of CFTR to characterise ABCG2.
The team first genetically engineered several standard mammalian cell types to make regular or mutant versions of ABCG2. Cells with the mutated ABCG2 gene contained much less of the ABCG2 protein than cells making the regular form. Additionally, the researchers found that the mutation made it difficult for ABCG2 molecules to get to their proper place on the cell surface. Since ABCG2 pumps molecules from the inside of the cell to the outside, it is not functional anywhere but the cell surface.
The team then lowered the temperature at which the ABCG2-making cells were growing, and found more mutant ABCG2 at the cell surface. Guggino says this finding suggested that the lower temperature had stabilised ABCG2 and helped it achieve its proper 3-D conformation, because proteins that don’t assume the right shape are likely to be broken into pieces for reuse, preventing them from reaching their final destinations.
When ABCG2 and CFTR are lined up, their structures are very similar. In fact, one of the most common cystic fibrosis mutations, a CFTR deletion of amino acid F508, lines up next to the Q141K mutation in ABCG2 and causes similar results in the protein’s location and processing.
Knowing that the F508 deletion in CFTR creates instability in a certain part of the protein, the researchers introduced additional mutations intended to stabilise the wobbly region of the Q141K mutant ABCG2. As predicted, they found that this stabilisation increased the amount of ABCG2 on the cell surface, suggesting again that ABCG2 had been saved from the recycling bin.
To confirm the involvement of the recycling process, the team fed the cells several small molecules known to help malformed proteins avoid degradation. One molecule, VRT-325, partially restores CFTR’s activity. The same molecule was also able to increase the amount of mutant ABCG2 found in the cells and on their surfaces, and to decrease the amount of uric acid in the cells, bringing it within the normal range.
‘Though there are many more lab tests needed before clinical trials can even be designed, our results represent an important step forward in both understanding how gout results from this mutation and finding a treatment,’ says Guggino. John Hopkins Medicine