High sensitivity high throughput biosensor measures metabolite levels that indicate disease

Researchers at the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, develop a biosensor that improves sensitivity to 1-methylnicotinamide (1-MNA) in urine by orders of magnitude without the need for sample purification.

Metabolites are a common indicator of disease. In particular 1-MNA levels are increased by cancer, liver disease, obesity and metabolic disease. At present mass spectrometry and nuclear magnetic resonance measurements are commonly used to measure quantities of metabolites in samples but they are expensive and complicated limiting their widespread use. Instead Masaya Ueno, Tomoki Ogoshi and Atsushi Hirao at Kanazawa WPI-NanoLSI use a type of pillararene molecule (Figs. 1 and  2) as a biosensor, getting around the need for high purification of other 1-MNA biosensing molecules while improving sensitivity and specificity.

1-MNA is produced in the body through the methylation of nicotinamide (Nam) by nicotinamide N-methyltransferase (NNMT) during the metabolism of the vitamin B3 vitamer niacin. As such it provides a measure of NNMT activity, which is elevated in some cancers. There is evidence that 1-MNA levels correlate with the how aggressive the tumour is and suppressing the gene for NNMT diminished certain behavioral symptoms of disease. As such the researchers explain in their report “monitoring the NNMT expression and activity in patients by quantification of 1-MNA is important for elucidating and diagnosing their pathology.”

Previously they had demonstrated some potential for the molecule pillar[6]arene functionalized with 12 carboxylate anions (P6AC) as a 1-MNA sensor, since it binds to 1-MNA and inhibits fluorescence due to photo-induced electron transfer. However extensive sample purification was needed and even then, although the millimolar concentrations in murine urine could be detected, the P6AC biosensor lacked the sensitivity to detect the micromolar concentrations found in culture supernatants of human cancer cells.

To find a biosensor with better sensitivity, the researchers investigated the binding between 1-MNA and pillar[6]arene functionalized with sulfonate groups (P6AS). They found that the binding affinity was 700 times greater than for P6AC, leading to a biosensor with sub micromolar sensitivity, even in unpurified human urine, although detection of the higher concentrations in mouse murine was better. However the researchers noted that detection in human serum was not possible due to the higher levels of autofluorescence.

By comparison the detection sensitivity possible with mass spectrometry is nanomolar but the throughput is much lower. The researchers suggest that the high throughput of the P6AS biosensor could make it suitable for screening 1000s of potential NNMT inhibitors, which may help towards a treatment for diseases like liver disease and cancer.

The researchers explain the higher sensitivity thanks to the stronger acidity of sulfonate groups compared to carboxylate groups. They conclude their report of the work, “Further improvement of our strategy will contribute to high-throughput screening of NNMT inhibitors, diagnosis of liver diseases, and imaging of human cancer cells in vivo.”

Glossary

Pillar[n]arenes

The pillar[n]arenes take their names from their pillar shapes and macrocyclic structures. The researchers were prompted to investigate the potential of P[6]AS as a 1-MNA detector following previous work by another group demonstrating the high binding affinity of P[n]AS for ammonium ions.

Binding affinity

The binding affinity refers to the strength of bond between the ligands on the protein and the candidate molecule, typically through intermolecular interactions such as ionic or hydrogen bonding or van der waals forces. The is a higher binding affinity when the attractive forces are higher.

Mass spectrometry

Mass spectrometers identify molecules based on their mass. The molecule is ionized and then accelerated through a field. The trajectory the molecule takes gives an indication of the charge mass ratio of the ion.

 

Funding acknowledgments

M.U. was supported by a Grant-in-Aid for Scientific Research (C) (23K06672) from the Ministry of Education, Culture, Sports, Science, and Technology, (MEXT), Japan. A.H. was supported by a Grant-in-Aid for Scientific Research (A) (19H01033) from MEXT and a Project for Cancer Research and Therapeutic Evolution (P-CREATE) (19cm0106104h0004) from the Japan Agency for Medical Research and Development (AMED). T.O. was supported by the CREST program of the Japan Science and Technology (JST) agency (JPMJCR18R3) and a Grant-in-Aid for Scientific Research (A) (22H00334) from MEXT. This work was supported by a WPI-NanoLSI Transdisciplinary Research Grant from Kanazawa University. NanoLSI is supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan. This work was supported by WISE Program for Nano-Precision Medicine, Science, and Technology of Kanazawa University by MEXT. This work was also supported by MEXT Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence (CURE) Program (JPMXP1323015484).

About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University

Understanding nanoscale mechanisms of life phenomena by exploring “uncharted nano-realms”.

Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale.

https://nanolsi.kanazawa-u.ac.jp/en/

About the World Premier International Research Center Initiative (WPI)

The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS).

See the latest research news from the centers at the WPI News Portal: https://www.eurekalert.org/newsportal/WPI

Main WPI program site: www.jsps.go.jp/english/e-toplevel

About Kanazawa University

As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities.

The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas.

http://www.kanazawa-u.ac.jp/en/

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