Nano Crystals for mega Fluorescence Amplification

SuperNova in the test tube

von Prof Dr Reinhard Renneberg, Jan Engels, Dr Hans-Georg Eisenwiener

We tell the exciting story of how mega ­fluorescence amplification was ­invented. ­
First, there was the question: How can ­previous biochemical evidence reactions ­be ­amplified million-fold to detect the bio­­chemical “needles in a haystack” (substances ­at very low concentrations) in a multicomponent system like a blood sample?

A mega idea supplied a possible solution based on the following idea: If a nano crystal, comprising of millions of potentially fluorescing molecules, is used as label (“marker”) in immune-chemical antigen-antibody tests or hybridisation reactions (DNA tests) instead of one or a few singular fluorescing molecules, calculations suggest a potential million-fold stronger fluorescence and thus amplification. If this kind of nano crystal is dissolved quickly and then triggered to fluorescence by a chemical reaction – hydrolysis –, fluorescence of millions of potentially fluorescing molecules will suddenly occur. This can be compared to a “SuperNova explosion in space”. The fluorescing molecule found and chosen was fluoresceindiacetate (FDA), a non-fluorescing organic molecule that fluoresces after hydrolysis.

A lack of detectability and quantifiability of a substance in a ­complex sample matrix is a recurring problem.

Where are highly sensitive detection reagents needed?

The proof of an analyte in the blood, serum/plasma, urine, CSF or saliva is “tedious and stressful” or not possible at all yet - particularly when DNA, antigens, antibodies or other substances are to be documented that are only present in very low concentrations (e.g. in the nano, pico or femto gram/mL range) or if it is difficult or painful to acquire a larger amount of sample material.
With our current determination methods – using colloidal gold or classic fluorescence markers – cardiac markers such as Troponin I/T, FABP or BNP/NT-proBNP can be determined in quality and quantity using 50–100µL capillary blood. How to get 50–100µL of capillary blood from the fingertip, though? This is not without pain. The super label FDA as a nano crystal does not require any more than 5–10µL of blood. This amount is easy to acquire.

Heureca in the night lab

The FDA story

In 1999/2000, Dieter Trau, at that time professor at the National University Singapore (NUS), was working on his doctoral thesis at the Hong Kong University of Science and Technology (HKUST) in the lab of Prof. Dr Renneberg. His research area was layer-by-layer encapsulation of different enzymes using poly-electrolytes. Among others, he also used the organic substance fluoresceindiacetat (FDA), which is commercially provided in crystalline form.

When Dieter Trau achieved polyelectrolyte labelling of large FDA crystals, he had an idea for how to continue using his previous experiments directly: “Why not use a fluorescing crystal for fluorescence testing instead of the usual evidence methods where only a few fluorescing molecules will adhere to every antibody?”

Dieter Trau labelled finely ground FDA crystals first with polyelectrolyte layers of PAH (polyallylamine hydrochloride) and PSS (polystyrene sulfonate). Then he attached antibodies to them to try out an immune test. After various tests and experiments, he was able to successfully label an FDA crystal with antibodies and then bind antigens to it. However, a reaction of non-fluorescing FDA into strongly fluorescing fluorescein was missing to produce the signal. For this, Trau first used enzymes (esterases) that are also used in cell cultivation. However, they react relatively slowly, which weakened the strong fluorescence effect again. Further tests led to hydrolysis of the acetate groups by highly concentrated sodium hydroxide (NaOH) into acetic acid and uranin (Fig. 1).

FDA is, however, an organic compound. They do not dissolve well in water, but very well in organic solvents, such as DMSO or IPA (isopropanol alcohol). With the addition of a “releasing ­reagent” comprising of, e.g., DMSO and sodium hydroxide, the crystals “exploded” like a super nova and the entire liquid suddenly turned into a strongly greenly fluorescing solution that can be seen by the unaided eye (Fig.2). The basis for the “SuperNova fluorescence” is the immediate dissolution of the crystal, which then permits the quick chemical reaction between the caustic soda and the FDA. Prof. Renneberg enthusiastically called out “Super!” and Dieter Trau added “And new is nova”. This was how the name – “SuperNova” – was coined. It was patented not long after [1].

“Wonder substance” FDA

The prerequisites: It should be a compound that is present in ­crystalline form, well-specified and not fluorescent as such. It must be possible to grind or recrystallise the crystal down to nanometre size. Why crystals? They contain the tightest packing of molecules in nature. After the crystal had been dissolved by a “reaction mixture”, the dissolved molecules instantly continued to react and all dissolved molecules fluoresced. Dieter Trau thus discovered in Hong Kong that fluoresceindiacetate (FDA) was a mega fluorescence amplifier [2].

Fluoresceindiacetat (FDA) is a derivative of the known fluorescing molecule fluorescein, which is often used in immunoassays. The two substances differ molecularly by two acetate groups attached to the FDA. FDA as such has nearly no fluorescence at all and is mainly used for bacteria and cell cultivation. FDA reacts with hydrolysis catalysing enzymes (specifically esterases) to fluorescein because the two acetate groups are split off. This is reflected in the intense green colour and fluorescence that can be seen well in the living cells using a fluorescence microscope. This reaction permits differentiation between living and dead cells, since dead cells no longer produce any enzymes.

Enormous amplification potential

A single FDA crystal ball 100?nm is size holds an unbelievable 1,200,000 (1.2 M) FDA molecules. Comparing the roughly 1.2 M molecules in the crystal used to roughly ten molecules directly bound to antibodies, there is a ratio of 1:120,000. This is, of course, reflected in the fluorescence strength as well.

Core question: How can a better or stronger signal or - more precisely - a higher signal/noise ratio be produced? Wouldn't it be better to use a nano crystal with ­millions of FDA molecules instead of a few fluorescing molecules of, e.g., FITC, to achieve the analysis sensitivity, characterised by the 3 NCCLS-parameters – limit of detection, limit of determination and limit of quantification – in the ng/pg- to fg-area for small available samples?

In contrast to today's “state-of-the-art” procedures, this is possible by not only binding molecules of fluorescence labels to antigens, antibodies or DNA, but by nano crystals of fluorescing molecules being bound instead. This means: Instead of the roughly ten fluorescing molecules that are covalently bound to antibodies, the reverse binding order uses a complete crystal comprising of a derivative of a fluorescing ­molecule to which the antibodies are bound directly. This bond between the crystal and the antigen or antibody can be adsorptive or covalent in origin. The strength of the bond can be increased by preliminary coating of the crystal with charged polyelectrolyte ­layers, the layer-by-layer encapsulation [3].

The great challenge: Competition for the PCR?

In addition to optimisation of particle size, immune assays for different antigens and antibodies with a lower limit of determination than conventional assets are being developed as well [4]. Direct evidence for just a few DNA molecules would be even more attractive. The current standard is – next to several other amplification methods - the polymerase chain reaction that leads to about 1 M DNA copies per hour. It is the unchallenged state of the art.

How close would the AmpCrystal (SuperNova)-method come to the range of DNA-PCR amplification? How many DNA molecules would be required for detection? What would be possible with SuperNova without cyclers and expensive enzymes?

The method: A nano crystal is not coated with antibodies but with single-stranded DNA. They bind to complementary DNA from a sample that is bound to a catcher DNA in a first step. It can be detected after addition of the releasing reagent and connected mega fluorescence generation [5]. It would be possible to combine the AmpCrystal method with PCR and thus save cycles - or to replace the PCR in a few tests (e.g. in microbiology).

Bibliography
[1] Trau D. et al. (2004) US 20040014073
[2] Trau D. et al. (2002) Anal. Chem. 202, 74, 5480?–5486
[3] Caruso F. et al. (2000) Langmuir, 16, 1485?–1488
[4] Chan CPY. et al. (2008) Adv Biochem Engin/Biotechnol 109:123–154
[5] Chan CPY. et al. (2007) Analytica Chimica Acta 584 7?–11

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