Final Report Summary - MRI_NANOBIOSENSOR (Novel Nanobiosensors for Identifying Microorganisms Using Magnetic Relaxation Switches)
Project context and objectives
The objectives are to develop sensitive, robust and versatile sensors for nucleic acids in point-of-care or in-the-field-type assays that can be used for the detection of microorganisms in third-world water supplies, food products or medical applications such as the bedside identification of blood-borne organisms that cause sepsis. The advantages of magnetic relaxation switch (MRS) methodology for point-of-care assays include its use of radiofrequency radiation rather than light (indifference to light-based interferences), and its use of solution-phase chemistry (no solid phase, no separation of free and bound as in enzyme-linked immunosorbent assays - ELISAs). The current state-of-the-art methods of identifying microorganisms are typically microscopy-based or culture-based and are not readily adaptable to such point-of-care uses.
Work performed
The research was divided into two phases:
- Phase I provided fundamentally new methods for the rapid detection of nucleic acids with applications to microorganism detection using MRS methods;
- Phase II provided the proof-of-concept for the application of MRS within a new tumoral cell-line detection in vitro system.
The proposed research is highly interdisciplinary, joining concepts of nanoparticle synthesis, surface chemistry, magnetic resonance and nucleic acid sequences to define the family of the microorganism.
In the field of detecting and quantifying nucleic acids there are two large research areas:
- non-amplification methods that include techniques like fluorescence in situ hybridisation or Northern blot;
- amplification-based methodologies that include techniques like reverse transcription polymerase chain reaction - rt-PCR - or nucleic acid sequence-based amplification.
In the first period of Phase I (2010), our approach was the identification of microorganisms based on ribosomal ribonucleic acid (rRNA) detection using a non-amplification methodology. The rRNA sequence was chosen for the following reasons:
- it is present in all organisms;
- it is the most abundant nucleic acid in bacteria;
- it can be detected without amplification methods;
- it contains highly conserved sequences and hyper-variables sequences that allow us to design general and species-specific probes;
- free databases exist with specific probes targeting rRNA.
In first year of research we validated the MRS technology as a non-amplification technique and our efforts were focused on the first three objectives that were described in the grant proposal (Part B).
In the second period of Phase I (2011) our research was focused on developing a new amplification-based methodology for the ultrasensitive detection of nucleic acids. First results showed much potential in this new area, which will expand the sensibility of nucleic acids and microorganism detection using the MRS technology.
Main results
Phase I: The project has proved, for the first time, the application of MRS methodology for identifying rRNA and, hence, microorganism detection using a non-amplified technique. Based on Fuchs' work, we selected an 18-base oligonucleotide sequence as our target molecule for the switch, the one that Fuchs identified as 100 % accessible. The detection by MRS was achieved by using superparamagnetic nanoparticles that were functionalised with complementary oligonucleotides.
The switch showed a very good response with ?T2 values over 40 ms (?T2 = /T2 initial - T2 final/) that could be tuned by adjusting the initial concentration of the reactants. Incubation with a non-complementary strand did not produce any signal showing that our MRS was selective towards the oligonucleotide sequences.
By applying the magnetic field-enhanced target aggregation technique to our switch, the sensitivity was increased to nearly double when applied sequentially to a normal MRS technique. It was also discovered that the MRS system was extremely sensitive to buffer changes and both ?T2 values and selectivity were dramatically affected when we moved to other buffers.
The project has also bridged the gap for a new ultrasensitive MR sensor of nucleic acids in amplification methodologies (PCR, rt-PCR, etc.) with a wide range of applications in molecular biology and biochemistry. The need for sequence-independent PCR detection methods has led to the search for fluorochromes that bind tightly to double-stranded deoxyribonucleic acid (dsDNA), with ever greater increases in fluorescence upon binding. Our approximation to this problem has been the attachment of a fluorochrome to the surface of a superparamagnetic nanoparticle to obtain DNA-binding fluorochrome-magnetic nanoparticles. The new sensor showed a slightly higher sensibility when used to monitor a model PCR reaction by fluorescence. When monitored by magnetic resonance (MR), the sensitivity of the sensor was up to ten times higher than the registration by a standard method based on fluorescence.
Phase II: We developed a new proof-of-concept application of MRS, termed a surface-mediated magnetic relaxation switch (SM-MRS), for the in vitro detection of tumoral cell lines through its abnormal glycosylation patterns and specific lectin-functionalised nanoparticles (NPs) (or vice versa). Preliminary experiments with carbohydrate-functionalised magnetic NPs and different lectin-decorated surfaces (glass and polymer) were performed and analysed by MRI and relaxometry. The SM-MRS showed a good response and selectivity towards selected carbohydrates, which may be easily translated to point-of-care devices. Cell experiments to determine the response of carbohydrate-decorated NPs against cancer cell lines within our platform were performed but reported limited or zero sensitivity on the conditions studied. Further experiments are ongoing to validate both the application and the proof-of-concept defined above.
Socioeconomic impact of the project
The project has succeeded in both phases: in Phase I, designing nucleic acid biosensors for both amplifying and non-amplifying methods and in Phase II, the design of a new platform with a potential application on tumoral cell detection.
Phase I: For the non-amplifying method, a stable nanoparticle platform that is able to detect rRNA from E. Coli was obtained. The measurements were accurate and fast but sensitive to external factors like ionic strength and the type of buffer used. These factors limit the clinical applications at present.
For the amplifying method, success was achieved by combining three novel principles:
- fluorochrome-mediated, DNA-binding surface design;
- multivalency-enhanced, solution-phase, microaggregate formation when fluorochrome-functionalized NPs bind DNA;
- detection of NP/DNA microaggregates by MR.
These three principles were combined to obtain a highly sensitive general method of detecting the DNA generated by the PCR reaction, without the risk of post-amplification contamination. This general method can be applied to basic research and translated to clinics (clinical microbiology, virology, haematology, etc.). A patent covering these applications has already been filed and recently licensed to the company T2-Biosystems.
Phase II: A new analytical platform, the so-called surface-mediated magnetic relaxation switch (SM-MRS), has been designed and tested with carbohydrate-functionalised NPs. The preliminary results showed a good response with proteins and might offer several potential applications in biomedicine, such as array cancer screening.
The objectives are to develop sensitive, robust and versatile sensors for nucleic acids in point-of-care or in-the-field-type assays that can be used for the detection of microorganisms in third-world water supplies, food products or medical applications such as the bedside identification of blood-borne organisms that cause sepsis. The advantages of magnetic relaxation switch (MRS) methodology for point-of-care assays include its use of radiofrequency radiation rather than light (indifference to light-based interferences), and its use of solution-phase chemistry (no solid phase, no separation of free and bound as in enzyme-linked immunosorbent assays - ELISAs). The current state-of-the-art methods of identifying microorganisms are typically microscopy-based or culture-based and are not readily adaptable to such point-of-care uses.
Work performed
The research was divided into two phases:
- Phase I provided fundamentally new methods for the rapid detection of nucleic acids with applications to microorganism detection using MRS methods;
- Phase II provided the proof-of-concept for the application of MRS within a new tumoral cell-line detection in vitro system.
The proposed research is highly interdisciplinary, joining concepts of nanoparticle synthesis, surface chemistry, magnetic resonance and nucleic acid sequences to define the family of the microorganism.
In the field of detecting and quantifying nucleic acids there are two large research areas:
- non-amplification methods that include techniques like fluorescence in situ hybridisation or Northern blot;
- amplification-based methodologies that include techniques like reverse transcription polymerase chain reaction - rt-PCR - or nucleic acid sequence-based amplification.
In the first period of Phase I (2010), our approach was the identification of microorganisms based on ribosomal ribonucleic acid (rRNA) detection using a non-amplification methodology. The rRNA sequence was chosen for the following reasons:
- it is present in all organisms;
- it is the most abundant nucleic acid in bacteria;
- it can be detected without amplification methods;
- it contains highly conserved sequences and hyper-variables sequences that allow us to design general and species-specific probes;
- free databases exist with specific probes targeting rRNA.
In first year of research we validated the MRS technology as a non-amplification technique and our efforts were focused on the first three objectives that were described in the grant proposal (Part B).
In the second period of Phase I (2011) our research was focused on developing a new amplification-based methodology for the ultrasensitive detection of nucleic acids. First results showed much potential in this new area, which will expand the sensibility of nucleic acids and microorganism detection using the MRS technology.
Main results
Phase I: The project has proved, for the first time, the application of MRS methodology for identifying rRNA and, hence, microorganism detection using a non-amplified technique. Based on Fuchs' work, we selected an 18-base oligonucleotide sequence as our target molecule for the switch, the one that Fuchs identified as 100 % accessible. The detection by MRS was achieved by using superparamagnetic nanoparticles that were functionalised with complementary oligonucleotides.
The switch showed a very good response with ?T2 values over 40 ms (?T2 = /T2 initial - T2 final/) that could be tuned by adjusting the initial concentration of the reactants. Incubation with a non-complementary strand did not produce any signal showing that our MRS was selective towards the oligonucleotide sequences.
By applying the magnetic field-enhanced target aggregation technique to our switch, the sensitivity was increased to nearly double when applied sequentially to a normal MRS technique. It was also discovered that the MRS system was extremely sensitive to buffer changes and both ?T2 values and selectivity were dramatically affected when we moved to other buffers.
The project has also bridged the gap for a new ultrasensitive MR sensor of nucleic acids in amplification methodologies (PCR, rt-PCR, etc.) with a wide range of applications in molecular biology and biochemistry. The need for sequence-independent PCR detection methods has led to the search for fluorochromes that bind tightly to double-stranded deoxyribonucleic acid (dsDNA), with ever greater increases in fluorescence upon binding. Our approximation to this problem has been the attachment of a fluorochrome to the surface of a superparamagnetic nanoparticle to obtain DNA-binding fluorochrome-magnetic nanoparticles. The new sensor showed a slightly higher sensibility when used to monitor a model PCR reaction by fluorescence. When monitored by magnetic resonance (MR), the sensitivity of the sensor was up to ten times higher than the registration by a standard method based on fluorescence.
Phase II: We developed a new proof-of-concept application of MRS, termed a surface-mediated magnetic relaxation switch (SM-MRS), for the in vitro detection of tumoral cell lines through its abnormal glycosylation patterns and specific lectin-functionalised nanoparticles (NPs) (or vice versa). Preliminary experiments with carbohydrate-functionalised magnetic NPs and different lectin-decorated surfaces (glass and polymer) were performed and analysed by MRI and relaxometry. The SM-MRS showed a good response and selectivity towards selected carbohydrates, which may be easily translated to point-of-care devices. Cell experiments to determine the response of carbohydrate-decorated NPs against cancer cell lines within our platform were performed but reported limited or zero sensitivity on the conditions studied. Further experiments are ongoing to validate both the application and the proof-of-concept defined above.
Socioeconomic impact of the project
The project has succeeded in both phases: in Phase I, designing nucleic acid biosensors for both amplifying and non-amplifying methods and in Phase II, the design of a new platform with a potential application on tumoral cell detection.
Phase I: For the non-amplifying method, a stable nanoparticle platform that is able to detect rRNA from E. Coli was obtained. The measurements were accurate and fast but sensitive to external factors like ionic strength and the type of buffer used. These factors limit the clinical applications at present.
For the amplifying method, success was achieved by combining three novel principles:
- fluorochrome-mediated, DNA-binding surface design;
- multivalency-enhanced, solution-phase, microaggregate formation when fluorochrome-functionalized NPs bind DNA;
- detection of NP/DNA microaggregates by MR.
These three principles were combined to obtain a highly sensitive general method of detecting the DNA generated by the PCR reaction, without the risk of post-amplification contamination. This general method can be applied to basic research and translated to clinics (clinical microbiology, virology, haematology, etc.). A patent covering these applications has already been filed and recently licensed to the company T2-Biosystems.
Phase II: A new analytical platform, the so-called surface-mediated magnetic relaxation switch (SM-MRS), has been designed and tested with carbohydrate-functionalised NPs. The preliminary results showed a good response with proteins and might offer several potential applications in biomedicine, such as array cancer screening.