Final Report Summary - MICRO UTI DIAG (Microfluidic assay for rapid multiplexed detection of bacterial urinary tract infections)
Rapid, sensitive, and simple to use tests, capable of detecting or ruling out a disease at the point of care, have the potential to revolutionize healthcare both in the developed and developing world. In particular, sensitive, sequence specific detection of nucleic acids can be used for early detection of infectious diseases (e.g. by detection of rRNA or genomic DNA, for early screening of cancer (e.g. by detection of miRNA or microsatellites), or genetic diseases. However, direct detection of these targets is fundamentally limited by their low concentrations and the associated slow hybridization kinetics of the target sequences and their probes, typically on the order of several hours.
PCR and other amplification methods are the gold standard in detection of nucleic acids, and overcome reaction rate limitations by creating additional copies of the target sequence, thus driving the hybridization reaction to completion. Amplification methods are by far the most sensitive, and can achieve a theoretical limit of detection as low as a single copy per ml. However, PCR reactions are time consuming, suffer from an inherent amplification bias, require significant sample preparation, and a well-controlled environment. At the same time, many practical applications do not require the sensitivity of a single copy. This results in a growing need for simpler and faster methods for sequence specific detection of nucleic acids, which are not based on amplification. Such applications include diagnosis of viral infections (such as Hepatitis B, Hepatitis C, respiratory syncytial, or cytomegalovirus), bacterial infections (such as urinary tract infections and pneumonia), and cancer screening (such as pancreatic cancer).
ITP is an electrophoretic separation and preconcentration technique. In peak mode ITP, analytes of interest are focused at the interface between a high electrophoretic mobility leading electrolyte (LE) and a low mobility trailing electrolyte (TE). The sample is typically mixed with the TE and an initial interface between the LE and TE is established. In order to focus, sample ions must have an intermediate electrophoretic mobility between those of the LE and TE. When an electric field is applied, such ions over-speed the slow trailing ions and accumulate at the migrating LE-TE interface, creating a highly concentrated zone.
In our work, we couple ITP based focusing of nucleic acids with PNA probes. PNA is an artificial DNA analogue in which the natural negatively charged deoxyribose phosphate backbone has been replaced by a synthetic neutral pseudo peptide backbone. The four natural nucleobases are retained on the backbone at equal spacing to the DNA bases. We inject the sample and a high concentration of fluorescently labeled PNA probes into the TE reservoir of an anionic ITP setup, allowing probes to rapidly bind to any matching target sequences present. The buffer system is chosen such that the electrophoretic mobility of the TE is higher than that of the free (unhybridized) probes but lower than that of the PNA-DNA hybrids. Therefore, once an electric field is applied, excess (unbound) PNA probes remain in the reservoir, while the negatively charged PNA-DNA hybrids electromigrate and focus at the ITP interface, resulting in a fluorescent signal. Hence, a fluorescent signal is obtained only in the presence of target sequences “carrying” the otherwise neutral PNA probes to the interface. This allows a highly sensitive, direct detection of target nucleic acids while completely eliminating background noise associated with unbound probes.
We showed that the use of PNA-based probes enables to completely eliminate the signal of unhybridized probes in the ITP interface, resulting in a LoD of 100 fM, and a dynamic range of 5 decades. The main advantage of the assay is in its simplicity, as it requires only a straight channel and two (specific) buffers. Moreover, we demonstrated that this assay can be successfully implemented for detection of nucleic acids directly form biological samples, without loss of signal.
We also developed two additional novel ITP based methods. One is a microfluidic assay for continuous and quantitative detection of whole bacteria, eliminating the need for additional preliminary lysis steps. The other is a new method for accurate detection of sample location in peak mode ITP. Altogether, these developments can be beneficial in a wide range of rapid diagnostics applications where detection of nucleic acids is required, including detection of genomic DNA, bacterial 16S rRNA and viral DNA.
http://microfluidics.technion.ac.il
PCR and other amplification methods are the gold standard in detection of nucleic acids, and overcome reaction rate limitations by creating additional copies of the target sequence, thus driving the hybridization reaction to completion. Amplification methods are by far the most sensitive, and can achieve a theoretical limit of detection as low as a single copy per ml. However, PCR reactions are time consuming, suffer from an inherent amplification bias, require significant sample preparation, and a well-controlled environment. At the same time, many practical applications do not require the sensitivity of a single copy. This results in a growing need for simpler and faster methods for sequence specific detection of nucleic acids, which are not based on amplification. Such applications include diagnosis of viral infections (such as Hepatitis B, Hepatitis C, respiratory syncytial, or cytomegalovirus), bacterial infections (such as urinary tract infections and pneumonia), and cancer screening (such as pancreatic cancer).
ITP is an electrophoretic separation and preconcentration technique. In peak mode ITP, analytes of interest are focused at the interface between a high electrophoretic mobility leading electrolyte (LE) and a low mobility trailing electrolyte (TE). The sample is typically mixed with the TE and an initial interface between the LE and TE is established. In order to focus, sample ions must have an intermediate electrophoretic mobility between those of the LE and TE. When an electric field is applied, such ions over-speed the slow trailing ions and accumulate at the migrating LE-TE interface, creating a highly concentrated zone.
In our work, we couple ITP based focusing of nucleic acids with PNA probes. PNA is an artificial DNA analogue in which the natural negatively charged deoxyribose phosphate backbone has been replaced by a synthetic neutral pseudo peptide backbone. The four natural nucleobases are retained on the backbone at equal spacing to the DNA bases. We inject the sample and a high concentration of fluorescently labeled PNA probes into the TE reservoir of an anionic ITP setup, allowing probes to rapidly bind to any matching target sequences present. The buffer system is chosen such that the electrophoretic mobility of the TE is higher than that of the free (unhybridized) probes but lower than that of the PNA-DNA hybrids. Therefore, once an electric field is applied, excess (unbound) PNA probes remain in the reservoir, while the negatively charged PNA-DNA hybrids electromigrate and focus at the ITP interface, resulting in a fluorescent signal. Hence, a fluorescent signal is obtained only in the presence of target sequences “carrying” the otherwise neutral PNA probes to the interface. This allows a highly sensitive, direct detection of target nucleic acids while completely eliminating background noise associated with unbound probes.
We showed that the use of PNA-based probes enables to completely eliminate the signal of unhybridized probes in the ITP interface, resulting in a LoD of 100 fM, and a dynamic range of 5 decades. The main advantage of the assay is in its simplicity, as it requires only a straight channel and two (specific) buffers. Moreover, we demonstrated that this assay can be successfully implemented for detection of nucleic acids directly form biological samples, without loss of signal.
We also developed two additional novel ITP based methods. One is a microfluidic assay for continuous and quantitative detection of whole bacteria, eliminating the need for additional preliminary lysis steps. The other is a new method for accurate detection of sample location in peak mode ITP. Altogether, these developments can be beneficial in a wide range of rapid diagnostics applications where detection of nucleic acids is required, including detection of genomic DNA, bacterial 16S rRNA and viral DNA.
http://microfluidics.technion.ac.il