Connector, marker and method for analysing biological samples
A connector is provided for analysing biological samples including at least one first affinity reagent configured to bind directly or indirectly to a target molecule, and a backbone connected to the first affinity reagent and having at least one first affinity interactor, wherein the first affinity interactor is configured to specifically bind to a second affinity interactor having a label in order to bind the label to the backbone, and wherein the backbone has a cleavage site for irreversibly separating the first affinity reagent and the first affinity interactor. In a further aspect, a marker and a method for analysing biological samples are provided.
1 . A set of markers for analysing biological samples, each marker comprising a connector and a label with a second affinity interactor, the connector comprising:
at least one first affinity reagent configured to bind indirectly to a target molecule,
a second affinity reagent bound to the first affinity reagent, wherein the second affinity reagent is configured to bind to the target molecule, wherein the second affinity reagent is an antibody, and the first affinity reagent is configured to bind to a fragment crystallisable (Fc) region of the antibody, and
a backbone connected to the first affinity reagent and comprising at least one first affinity interactor,
wherein the first affinity interactor is configured to specifically bind to a second affinity interactor comprising the label in order to bind the label to the backbone,
wherein the backbone comprises a cleavage site for irreversibly separating the first affinity reagent and the first affinity interactor, and
wherein antibodies of the markers of the set of markers are of a same isotype having the same Fc region that binds to the first affinity reagent, and wherein the antibodies are specific to different target molecules.
2 . The set of markers according to claim 1 , wherein the first affinity reagent is a nanobody, or an aptamer.
3 . The set of markers according to claim 1 , wherein the backbone comprises an oligonucleotide or a peptide.
4 . The set of markers according to claim 1 , wherein the cleavage site is a photocleavable cleavage site.
5 . The set of markers according to claim 1 , wherein the backbone is cleavable specifically at the cleavage site by an enzyme, cleavage light, or by a temperature change.
6 . The set of markers according to claim 1 , wherein the first affinity interactor and the second affinity interactor are configured to form a bioconjugate.
7 . The set of markers according to claim 1 , wherein the first affinity interactor is one of biotin or streptavidin, and the second affinity interactor is the other one of biotin or streptavidin.
8 . The set of markers according to claim 1 , wherein the connector comprises a plurality of first affinity interactors for binding a plurality of second affinity interactors.
9 . The set of markers according to claim 1 , wherein the label comprises at least a first fluorophore.
10 . A method for analysing a biological sample comprising the following steps:
a) providing at least a first set of markers, including the set of markers according to claim 1 , wherein the first set of markers comprises at least a first plurality of connectors configured to bind directly or indirectly to a first target molecule, and each first connector comprising a first label,
b) introducing at least the first set of markers into the sample in order for the markers to bind to their respective target molecule in the sample,
c) directing excitation light onto the biological sample, the excitation light being configured to visualise at least the first label, and
d) generating at least one optical readout from light emitted by at least the first label.
11 . The method according to claim 10 , wherein the first set of markers further comprises at least a second plurality of connectors configured to bind directly or indirectly to a second target molecule, and each second connector comprising a second label.
12 . The method according to claim 11 , wherein the first label and the second label have different fluorescent properties.
13 . The method according to claim 10 , wherein in a step e) cleavage site of the connectors of at least the first set of markers is cleaved.
14 . The method according to claim 13 , wherein in a step f) cleaved off second affinity interactor is removed.
15 . The method according to claim 13 , wherein the steps a) to d) are repeated with a second set of markers, wherein the second set of markers comprises at least a third plurality of connectors configured to bind directly or indirectly to a third target molecule, and each connector comprising a third label.