Hunor Sántha
5.3 CONSIDERATIONS FOR WATER SAMPLES AND IN-LINE (QUASI CONTINUOUS) OPERATION(QUASI CONTINUOUS) OPERATION
In the title of this chapter the word“sustainable”refers to the intention, that preferably and ideally no consumables should be used for the operation of the DINAMICS pathogen detection system, thus, this is true for the nucleotide release submodule too. If one considers, that in order for being selective in the detection our system deploys biosensors, which type of sensors always have only a limited lifetime/usability, the“no consumables”requirement is immediately handled with flexibility. Practically
Detection of Pathogens in Water Using Micro and Nano-Technology 72
speaking the exchange interval of the biosensor elements should be maximised, and the usage of single use/disposable consumables (e.g. filters, reagents etc.) should be minimised.
The requirements related to the “in-line” operation of the whole assay (and also of the sample preprocessing i.e. the nucleotide release) means, that the water supply chain should be monitored continuously (or at least quasi continuously, with a controlled delay) without human intervention. Alone from theoretical considerations one can deduce, that only the quasi continuous approach may work, since it is an axiom, that the affinity type biosensors all need a certain (tailorable but predetermined) incubation time for exact concentration measurements. It means, that depending on the complete assay time vs. the acceptable maximum delay in detection of a pathogen hazard, multiple “pipelines”have to be operated simultaneously with shifted cycle-starts.
Under the aegis of the DINAMICS project a“reagentless cell lysis and DNA/RNA release method” and prototype module was targeted as the part of the final prototype device. However at last the meaning of“reagentless”became limited to anything other than the enzymatic cell wall/cell membrane digestion based methods, which would be too time consuming. Two partners with microbiological background–MikroMikoMed Ltd. (Budapest, Hungary) and Water Research Institute, (Bratislava, Slovakia)–and one partner with hardware and software technological background–Budapest University of Technology and Economics–Dept. for Electronics Technology (Budapest, Hungary)–were involved in this RTD work.
After the above described general technical considerations the target organisms for the final prototype have been identified from groups of bacteria, viruses and parasites covering more than ten taxons. The outcome is presented in Table 5.2 below. The reasoning behind this set of choice can be found in a report of the consortiums work entitled “Target Contaminant Selection”. Also included in the table and among the functional requirements are the detection limits (or device sensitivity) for each organism, described in terms of the number of organisms in the daily consumed quantity of water, estimated at 2 litres per person per day.
As discussed in more detail in the “User Requirements Specification”report of the consortium, the detection limits provided in this table are a simplified representation of a complex issue. Their expression in terms of water consumed downstream as distinct from water sampled upstream implies the need to estimate dispersion.
It worth noting, that the method of filtering/concentration of pathogen microbes from the water (i.e.
centrifugation or filtering) has strong implications on the applicable DNA/RNA release methods too.
The initial hypothesis was that a one- or two-stage filtering will be necessary either way, because the microbiological standards rely on filters with different pore sizes. The main question was if the cell lysis/nucleotid release should take place alreadyin-situin the filter (ConceptB)) or only in a subsequent reaction chamber with freely dispensed/floating microbes after a wash-out from the filter (ConceptA)).
Concept (A)
A separate microfluidic module impacts on a highly concentrated solution of freely moving cells which are dispersed in a buffer solution after removal of pathogen cells from the filter(s). The procedure and its results supposed to be observable through optical microscope. A sketch of the method can be seen in Figure 5.4.
Concept (B)
Lysisin situin the filter membranes by means of an electrode pair pressed on the two sides of the filter membrane (the filter has to be immersed in a reagent before DNA/RNA content can be forwarded into the PCR module). This concept is based on a filter membrane with a pore diameter smaller than the
sample cell diameter, but larger than the obsolete components of the sample reservoir. Thus, the method consists of multiple subsequent steps. In the first step filters with a pore diameter appropriate for the largest cells should be applied, then a smaller pore sized filter, and so on. This way, in every step separate types of microbes can be disrupted, and their DNA/RNA prepared for further analysis.
Figure 5.5 shows, how it works.
Table 5.2 Target organisms and detection limits for the prototype aimed at in the DINAMICS project.
Category1 Kingdom Organism Type4 Priority2 Safety level3
Infectious Dose
Detection Limit
A Prokaryotes Yersinia pestis Gram (−) 1 3,2* .100
organisms
B Prokaryotes Shigellaspp. Gram (−) 1 2,3*** 10 organisms 10
B Prokaryotes Vibrio cholerae Gram (−) 1 2 103–106
A Virus Variola major dsDNA virus 2 N/A 10
Other Virus Norovirus,
Other Virus Rotavirus dsRNA virus 1 2 10–100 viral
particles
10
Other Virus Hepatitis A ssRNA virus 1 10–100 virus
particles
10
Other Virus Hepatitis E ssRNA virus 2 Unknown 10
B Eukaryotes Cryptosporidium parvum
1 2 One organism 10
Other Eukaryotes Giardia lamblia 1 2 One or more
cysts
10
*Y. Enterolytica, Y. Pseudotuberculosis
**S. Typhii
***Sh. Dyenteriae Typ1
1These categories have been taken from the U.S. Centers for Disease Control and Prevention (CDC)
2Priority refers to the priority within the DINAMICS project because of limited resources
3“Safety level”refers to CDC specified levels of precautions for Biological agent (1–4=lowest–highest)
4ss=single stranded, ds=double stranded
Detection of Pathogens in Water Using Micro and Nano-Technology 74
At last filtering proved to be a less integrable manner because of the huge challenge in automation of filter exchanges, thus ConceptB)was fallen off. ConceptA)relying exclusively on physical impacts became a first choice, but the possibilities were very diverse and none of the ways was really well established Figure 5.4 The schematic plan to implement a concept’A’lysis device.
Figure 5.5 The schematic of a concept’B’cell lysis device.
considering that the target pathogen list of table 2 contains more than 10 very-very different microbes, and the project demanded a 1) sustainable, 2) in-line, 3)“one-method-fits-all”solution.
Through various sources of information we concluded, that no commonly accepted guidelines exist in this area, thus, after a literature search revealing the state-of-the-art, certain in-house experiments, several rounds of conceptualization → hardware and software design a series of testing → evaluation → modification have been implemented.
The concept and the proof-of-concept experimental set-up has evolved from a simple purely DC electric field based cell lysis device across a triple-impact (i.e. simultaneous #1 heat, #2 ultrasonic waves, #3 AC electric field) concept towards the final, more industrial, viable and robust solution to be detailed in the last section of this chapter.