Sherlock Holmes in the blood: The trail of blue structures
Sometimes you don't need an electron microscope
Months ago, especially during the initial phase of using Novalytiv, we suddenly noticed a blue discoloration in the hydrogel structures. Unfortunately, I cannot see this under a dark field microscope—dark blue on black remains black. With my additional bright field condenser, I can now see it too, and it is still there.
So where does this come from?
Let’s start with the hydrogels themselves. It wasn’t just my remote viewing sessions that pointed to snakes or spider components. They can also be found in scientific documentation.
Spider silk/spider proteins are used as structural/scaffold material in hydrogels to produce “protein-based hydrogels” — i.e., water-rich polymer networks whose structural scaffold consists of spider protein.
https://news.ki.se/scientists-develop-gel-made-from-spider-silk-proteins-for-biomedical-applications
Such spider silk hydrogels can gel at 37 °C (i.e., body temperature) without the need for aggressive chemical crosslinkers. This makes them suitable as 3D scaffolds for cell cultures or drug delivery systems, or as biomimetic materials for tissue engineering. And this is exactly what I had suspected for some time based on the new forms appearing in the blood.
https://news.ki.se/recombinant-and-tuneable-spidroin-hydrogels-for-drug-release-and-cell-culture
A specific example: A hybrid gel consisting of hyaluronic acid (HA) and spider silk proteins (Ss) was successfully produced via chemical coupling (carboxyl to amino groups). The resulting hydrogel exhibits a stable porous structure, good water binding (swelling), and—depending on the composition—good mechanical properties and biocompatibility.
https://pmc.ncbi.nlm.nih.gov/articles/PMC8198725
Recent developments show that such spider silk hydrogels can be mechanically reinforced (e.g., by fiber reinforcement), making them interesting for softer tissue types.
https://www.researchgate.net/publication/395559160_Fiber_Reinforcement_of_Soft_Spider_Silk_Hydrogels
The same works with snake proteins
A prominent example is a hydrogel based on a synthetic, self-assembling nanofiber scaffold in which the snake proteins ecarin (procoagulant) and textilinin (antifibrinolytic) have been embedded.
The result is a “wound-sealing” gel that stops bleeding extremely quickly—in animal models, up to 60 seconds after application, even under blood-thinning conditions.
https://pubmed.ncbi.nlm.nih.gov/35652565/
This gel works precisely because the toxins are bound locally and do not have a systemic effect. The hydrogel thus acts as a carrier material that keeps the active proteins at the site of the injury.
The review paper on wound care with snake venom proteins points out that such approaches could open up a new class of hemostatics, especially for patients with coagulation problems or those taking anticoagulants.
https://www.drugdiscoverynews.com/could-snake-venom-usher-in-a-new-era-of-band-aid-15706
What does cross-linking with snake venoms or spider toxins mean?
In the case of snake toxins, it is usually not a matter of chemical cross-linking between polymer chains, but rather of using a hydrogel as a carrier matrix for active toxin proteins. The “cross-linking” therefore primarily consists of the hydrogel holding the toxin locally and releasing its effect — rather than structural cross-linking by the toxin itself.
In the case of spider toxins/spider silk proteins, on the other hand, these proteins are often used directly as structural components—in other words, the protein forms the framework of the hydrogel. This is done partly without external crosslinkers (self-gelling at body temperature) and partly through chemical coupling, e.g., between HA and spidroin. Here, crosslinking occurs in the classic sense of a polymer network.
And where does the BLUE come from?
Blue discoloration is possible when hydrogel cross-linking is dissolved, but only if certain chemical systems are present, such as copper-containing reactions, metal-protein complexes, or pH-dependent chromophores. This usually has nothing to do with snake or spider toxins directly.
BUT
Spider silk proteins (spidroins) contain functional groups that can form colored complexes with lanthanides.
Spider proteins contain tyrosine, histidine, serine, asparagine, carboxylates of the side chains, or carbonyl groups in the peptide backbone.
Many of these groups can coordinate Ln³⁺ ions.
So why does the color develop?
Lanthanides themselves absorb very weakly in the visible range (f-f transitions), but:
Protein-lanthanide complexes can develop visible colors (sometimes bluish to violet) through charge transfer, ligand field effects, and coordination to aromatic groups.
This is well documented experimentally for, e.g., Eu³⁺-protein complexes (reddish-purple, but also bluish tint with certain ligands) and Tb³⁺-protein complexes (reddish-purple, but also bluish tint with certain ligands).
This has been well documented experimentally for, e.g.:
Eu³⁺–protein complexes (reddish-violet, but also bluish tint with certain ligands)
Tb³⁺–protein complexes
Ce³⁺/Ce⁴⁺–complexes
Yb³⁺– and Tm³⁺–complexes
The color depends on the ligand field — proteins create different coordination environments than small organic ligands.
If a hydrogel contains spider silk protein and the cross-linking dissolves, free coordination sites suddenly become available and the lanthanide can form new complexes, making visible coloring possible.
Why does blue discoloration occur specifically when the cross-linking is dissolved?
When the polymer network in the gel breaks down, the ligands change. The metal ions detach from their original coordination sites and free amino acids/peptide fragments become mobile.
These can then form different colored Ln³⁺ complexes than in the solid gel.
If oxidation occurs in the presence of oxygen, some Ln ions, especially Der, can change their oxidation states and cause colored transitions to occur.
Changes in pH can alter the coordination chemistry.
All of these mechanisms can form new chromophores and thus exhibit blue discoloration.
But why BLUE?
Blue coloration is often observed in Ln-protein complexes when:
the lanthanide forms Ce³⁺, Yb³⁺, Tm³⁺ or special Eu/Tb complexes
the coordination produces “d-π feedback” or LMCT bands (ligand-→metal charge transfer)
There is absorption in the yellow-red range, so that visible light appears blue.
What does this mean specifically for a hydrogel?
If your hydrogel consists of PEG or spider silk protein and lanthanide ions, the following scenario occurs:
In an intact hydrogel, the lanthanide is firmly bound and the complex is therefore invisible.
When the cross-linking breaks down, new complexes form and peptide fragments are created. This results in a blue coloration, even a violet tint.
In other words, if you use a process that is capable of breaking down the cross-links in these hydrogels, they will show a blue discoloration under the microscope. In other words, successful dissolution!
If there are metal ions in the compound, you can test it by adding EDTA—if the color disappears again, this is proof of this. This would be proof that we are dealing with a lanthanide.
Summary
The blue coloration is caused by the formation of complexes between lanthanide metal ions and amino acid groups (especially aromatic and basic ones) in the spider proteins when the cross-links break down.
So what does the blue coloration on the gel indicate?
A blue coloration during breakdown means:
The cross-links have largely dissolved. The metal ions have been released. - Caution: binding agents are necessary to avoid overloading the liver and kidneys.
The protein fragments or spider silk motifs are mobile and re-coordinate. Partially oxidized or LMCT-active metal complexes are formed.
This is a clear indicator that the microstructure of the gel has changed.
And which lanthanide is most likely to cause BLUE STAINING?
Tm³⁺ shows a bright, almost fluorescent blue
Ce complexes tend to be darker blue/violet, sometimes gray-blue
Yb complexes have a bluish, slightly violet spectrum
In the case shown here, Ce is likely to be the most probable, as it exhibits a typical dark blue coloration.
Of all the lanthanides, cerium is the one that most frequently forms dark blue to blue-black complexes in the presence of proteins, peptides, or other organic ligands.
This is because cerium can switch between two oxidation states:
Ce³⁺ (colorless to slightly yellow) and Ce⁴⁺ (strongly oxidizing, forms colored LMCT complexes). When Ce³⁺ comes into contact with peptide fragments and oxygen, Ce³⁺/Ce⁴⁺ mixed complexes can form.
These have a ligand and the metal charge transfer bands, which appear dark blue to violet.
Spider silk proteins enhance this effect
The amino acids in spider silk (especially tyrosine, histidine, serine, carboxylates) are ideal ligands.
When the hydrogel cross-linking breaks down, these ligands are released and cerium re-coordinates, forming colored complexes.
Peptides from spider venom themselves can have an anesthetic effect. We are receiving more and more reports from people who say that they no longer feel minor injuries. But that will be the subject of a separate article.
