Nanomedicine (Lond). 2016 Aug;11(16):2185-95.

Electrochemical sensing of biomarker for diagnostics of bacteria-specific infections.

Alatraktchi FA1,2,3, Johansen HK3,4, Molin S2,3, Svendsen WE1.
  • 1Department of Micro- & Nanotechnology, Technical University of Denmark, Kgs-Lyngby, Denmark.
  • 2Department of Biotechnology & Biomedicine, Technical University of Denmark, Kgs-Lyngby, Denmark.
  • 3Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • 4Cystisk Fibrose Klinikken & Klinisk Mikrobiologisk Afdeling – Rigshospitalet, Copenhagen, Denmark.

Abstract 

Aim: Pseudomonas aeruginosa is a pathogen that is prevalent in serious infections in compromised patients worldwide. A unique virulence factor of this bacterium is the redox-active molecule pyocyanin, which is a potential biomarker for the identification of P. aeruginosa infections. Here we report a direct, selective and rapid detection technique of pyocyanin.

Materials & methods: Pyocyanin was detected by amperometry at a relatively high potential where the pyocyanin signal was unaffected by background contributions.

Results & conclusion: Pyocyanin was detected at concentrations down to 125 nM in a 50 μM mixture of interfering compounds with a reproducibility of r2 = 0.999 (n = 5) within 200 s. The results document a step toward a point-of-care technique for diagnosis of P. aeruginosa infections.

KEYWORDS:

Pseudomonas aeruginosa; amperometry; bacteria; biosensors; cystic fibrosis; detection; diagnosis; electrochemistry; electrodes; pyocyanin

PMID: 27464037; DOI: 10.2217/nnm-2016-0155
Supplement

Bacteria knows a trick to cheat the immune system. They communicate together and tell each other when they can start the virulent activity that makes you sick. They are in a kind of hibernation until they simultaneously decide that now is the time to break out an infection. The bacteria do this by sending out small signaling molecules that inform each other about the bacterial density. When they reach a certain threshold, all bacteria become dangerous. The bacteria can therefore initiate their invasion of the body perfectly synchronized.

The phenomenon is called Quorum sensing and is named after the Roman Senate where an assembly of decision makers was called a quorum. If there is not enough bacteria, the quorum sensing signal will disappear in the environment, but when they are enough, the signals will be accumulated and the bacteria will sense a change in the signal concentration in their environment. When the bacteria know they are sufficiently numerous, they will be able to trigger biochemical reactions that ensure their survival and cause damage to the human host.

In the clinic, body fluids such as sputum are used to determine if a patient is infected with bacteria in the lung. This is done via a microbiological culturing of a sputum sample to verify if pathogenic bacteria is present. Sputum cultures are specifically used to help identify the types of infections in the lungs and airways. Studies have shown that the environmental bacterium Pseudomonas aeruginosa can live up to a couple of years in a patient without being detected by conventional bacterial culturing.

We hypothesized that we can detect quorum sensing signals in sputum samples while they are still building up before the threshold where the infection breaks out. This way it will be possible to predict a bacterial infection before it establishes permanently. We targeted a quorum sensing dependent virulent factor called pyocyanin that is specifically excreted by P. aeruginosa.

Pyocyanin is an electroactive compound which means that it can undergo a redox-reaction when exposed to small potentials. We detected pyocyanin using electrochemical sensing, where a constant potential is applied to a sputum sample containing P. aeruginosa that secrete pyocyanin. The obtained current during the measurement is corresponding to the pyocyanin concentration existing in the sample. The method requires no sample pretreatment, no bacterial culturing and is capable of detecting pyocyanin in the nano-molar range within few minutes.

The main challenge of this method is that pyocyanin is excreted in a complex environment where a lot of other metabolites are released and where the background itself consists of many chemicals that could interfere with the target signal. We have developed a procedure for detecting nano-scale concentrations of pyocyanin in a complex background similar to what could be found in sputum. The main concept of this method is to find a small potential window where no other chemicals interfere with the signal of interest. This specific potential can then be used to detect pyocyanin. A higher current signal will be achieved with higher pyocyanin concentration while the background contribution will stay constant. This way it will be easy to filter out interference and translate the pyocyanin signal to actual concentrations.

This procedure makes it possible to study the bacterial virulent behavior and investigate whether the bacteria are on the threshold of starting an illness. If the sensors predict that a serious disease is on the way, the doctors can determine the most optimal treatment strategy, for example in the form of antibiotics. If antibiotics are given without any real indication, this can create resistance. But if the antibiotic treatment is given at the right time, it can save lives. This way we reduce the consumption of antibiotics and we prevent the infection from going out of control. Early diagnosis and treatment will consequently contribute to a longer and more convenient life for the infected patients.

 

 

Figure 1. Patient provides a sputum sample that is tested with the electrochemical sensor. The sensor can provide accurate values of the bacterial concentration in the sputum sample – even for very low concentrations.

 

The importance of this study

This work shows that it is possible to selectively detect very low concentrations of pyocyanin as a biomarker for the presence of the environmental bacteria P. aeruginosa. The detection was still possible after increasing the complexity of the background which suggests that this method can be used to accurately detect bacteria in human sputum. Early diagnosis of bacterial infections may allow targeted treatment strategies and thus significantly enhance the quality of life and life time expectancy of the patients. Because of the high sensitivity of our method we strongly believe that we can detect P. aeruginosa up to several years before they can be detected using conventional microbiological methods.

 

References

[1] Alatraktchi, F. A.-Z., Breum Andersen, S., Johansen, H. K., Molin, S., & Svendsen, W. E. (2016). Fast Selective Detection of Pyocyanin Using Cyclic Voltammetry. Sensors, 16(3).

[2] Alatraktchi, F. A.-Z., Johansen, H. K., Molin, S., & Svendsen, W. E. (2016). Electrochemical sensing of biomarker for diagnostics of bacteria-specific infections. Nanomedicine, 11(16), 2185–2195.

 

Acknowledgement

The authors sincerely acknowledge the Novo Nordisk Foundation Center for Biosustainability, Nanna Bild and Jesper Scheel for graphics and photography. Helle Krogh Johansen was funded by a clinical research stipend from The Novo Nordisk Foundation and Rigshospitalet Rammebevilling 2015-17 and Lundbeckfonden Grant R167-2013-15229.

 

Contact corresponding author:

Fatima AlZahra’a Alatraktchi, MSc

PhD student

Department of Micro-and Nanotechnology,

Department of Biotechnology and Biomedicine,

Novo Nordisk Foundation Center for Biosustainability

Technical University of Denmark

Faaat@nanotech.dtu.dk

 

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