Final Report Summary - ELENA (Electrochemical LEctin and glycan biochips integrated with NAnostructures)
The aim of the project was to develop novel ultrasensitive methods for analysis of complex sugars (glycans) with the aid of nanotechnology.
Importance of glycans
Glycans are present on the surface of 70-80% of all proteins in our body. Glycans can form much more complex structures compared to DNA and proteins and thus glycans are applied to code and store information. For example density of glycans can indicate the age of red blood cells for effective removal of old red blood cells from blood and division of blood into blood types (A, B, AB and 0) is according to the glycan type, which is attached to the red blood cell.
Glycans are heavily involved in many pathological processes such as virus entry, autoimmune diseases and cancer. HIV viruses have ability to enwrap themselves into a membrane of the host cell, covered by glycans, to stay hidden from immune system. Influenza viruses are categorised into subtypes such as H1N5 according to two types of proteins involved in recognition/processing of glycans. Autoimmune diseases and cancer are often associated with changed glycan composition, what can be used for diagnostic purposes.
Glycan analysis
One way is to use sophisticated/costly instruments requiring skilled operators and various pretreatment steps, which can complicate analysis. Our approach is to apply proteins designed by the nature to recognise glycans – lectins. Lectin biochips can detect glycans in an intact form (i.e. still attached to protein and even cells), thus providing a precious information about glycan, which can not be acquired by above-mentioned instruments.
In the project we focused on application of nanotechnology (nanomaterials or nanoscale controlled modification of surface by biomolecules) to develop various types of biochips/devices for glycan analysis. With application of nanomaterials with the size 10,000 lower than a diameter of a human hair or with controlled construction of biochips at nanoscale we can detect extremely low glycan concentration by factors ranging from million to billion in comparison to biochips based on other principles of analysis. At the same time we focused on development of biosensing techniques allowing to analyse complex samples such as human serum by modification of the surfaces with thin layers resisting non-specific binding. Thus, the biochips developed were very sensitive (thank to nanotechnology) and specific (thank to resisting layers) and ready for analysis in human serum.
Practical applications
Initially we developed devices with immobilised lectins (glycan recognising proteins) for glycoprofiling of serum samples from healthy individuals and patients suffering from rheumatoid arthritis or systemic sclerosis without any sample treatment (or using a moderate sample treatment). Such protocol allowed to distinguish these two set of samples, but only with a limited diagnostic potential.
Then we focused on glycoprofiling of particular glycoprotein present in serum samples such as PSA (prostate specific antigen), a biomarker of a prostate cancer (PCa). In this case antibody against PSA was immobilised on the device surface to selectively fish out PSA from serum sample and then lectins were applied to glycoprofile glycan on the PSA. This approach was utilised to see if the change of glycan present on PSA could be applied as novel biomarker to distinguish healthy individuals from PCa patients. Some lectins could provide diagnostically useful information and the detection method was integrated into clinically relevant format requiring a minute amount of blood for analysis in a time effective way. The PSA glycoprofiling approach is currently validated with a clinical partner with potential to be applied as a novel diagnostic (heathy vs. PCa) or prognostic (info about disease progression) PCa biomarker. A preliminary experiments suggest that similar approach could applied for novel breast cancer diagnostics or for diagnostics of chronic lymphocytic leukaemia.
In the inverted detection scheme glycans were immobilised on the surface of the biochips for detection of viral proteins, intact influenza viruses and antibodies against changed glycans present in the serum during cancer development and progression. This could be also applied for early cancer diagnostics.
Importance of glycans
Glycans are present on the surface of 70-80% of all proteins in our body. Glycans can form much more complex structures compared to DNA and proteins and thus glycans are applied to code and store information. For example density of glycans can indicate the age of red blood cells for effective removal of old red blood cells from blood and division of blood into blood types (A, B, AB and 0) is according to the glycan type, which is attached to the red blood cell.
Glycans are heavily involved in many pathological processes such as virus entry, autoimmune diseases and cancer. HIV viruses have ability to enwrap themselves into a membrane of the host cell, covered by glycans, to stay hidden from immune system. Influenza viruses are categorised into subtypes such as H1N5 according to two types of proteins involved in recognition/processing of glycans. Autoimmune diseases and cancer are often associated with changed glycan composition, what can be used for diagnostic purposes.
Glycan analysis
One way is to use sophisticated/costly instruments requiring skilled operators and various pretreatment steps, which can complicate analysis. Our approach is to apply proteins designed by the nature to recognise glycans – lectins. Lectin biochips can detect glycans in an intact form (i.e. still attached to protein and even cells), thus providing a precious information about glycan, which can not be acquired by above-mentioned instruments.
In the project we focused on application of nanotechnology (nanomaterials or nanoscale controlled modification of surface by biomolecules) to develop various types of biochips/devices for glycan analysis. With application of nanomaterials with the size 10,000 lower than a diameter of a human hair or with controlled construction of biochips at nanoscale we can detect extremely low glycan concentration by factors ranging from million to billion in comparison to biochips based on other principles of analysis. At the same time we focused on development of biosensing techniques allowing to analyse complex samples such as human serum by modification of the surfaces with thin layers resisting non-specific binding. Thus, the biochips developed were very sensitive (thank to nanotechnology) and specific (thank to resisting layers) and ready for analysis in human serum.
Practical applications
Initially we developed devices with immobilised lectins (glycan recognising proteins) for glycoprofiling of serum samples from healthy individuals and patients suffering from rheumatoid arthritis or systemic sclerosis without any sample treatment (or using a moderate sample treatment). Such protocol allowed to distinguish these two set of samples, but only with a limited diagnostic potential.
Then we focused on glycoprofiling of particular glycoprotein present in serum samples such as PSA (prostate specific antigen), a biomarker of a prostate cancer (PCa). In this case antibody against PSA was immobilised on the device surface to selectively fish out PSA from serum sample and then lectins were applied to glycoprofile glycan on the PSA. This approach was utilised to see if the change of glycan present on PSA could be applied as novel biomarker to distinguish healthy individuals from PCa patients. Some lectins could provide diagnostically useful information and the detection method was integrated into clinically relevant format requiring a minute amount of blood for analysis in a time effective way. The PSA glycoprofiling approach is currently validated with a clinical partner with potential to be applied as a novel diagnostic (heathy vs. PCa) or prognostic (info about disease progression) PCa biomarker. A preliminary experiments suggest that similar approach could applied for novel breast cancer diagnostics or for diagnostics of chronic lymphocytic leukaemia.
In the inverted detection scheme glycans were immobilised on the surface of the biochips for detection of viral proteins, intact influenza viruses and antibodies against changed glycans present in the serum during cancer development and progression. This could be also applied for early cancer diagnostics.