Multiplexed electrochemical liposomes applied to the detection of nucleic acids for Influenza A, Influenza B and SARS-CoV-2

Multiplexing is a relevant strategy for biosensors to improve accuracy and decision-making due to the increased amount of simultaneously obtained information. Liposomes offer unique benefits for label-based multiplexing since a variety of different marker molecules can be encapsulated, leading to intrinsic signal amplification and enabling a variety of detection formats. We successfully developed an electrochemical (EC) liposome-based platform technology for the simultaneous detection of at least three analytes by studying parameters to ensure specific and sensitive bioassay performance. Influenza A and B and SARS-CoV-2 sequences served as model system in a standard sandwich hybridization assay. Studies included encapsulants, probe distribution on liposomes and capture beads, assay setup and interferences between liposomes to also ensure a generalization of the platform. Ruthenium hexamine(III), potassium hexacyanoferrate(II) and m-carboxy luminol, when encapsulated separately into a liposome, provided desirable long-term stability of at least 12 months and no cross-signals between liposomes. Through the optimization process, low limits of detections of 1.6 nmol L−1, 125 pmol L−1 and 130 pmol L−1, respectively, were achieved in a multiplexed assay setup, which were similar to singleplex assays. Non-specific interactions were limited to 25.1%, 7.6% and 7.5%, respectively, through sequential liposome incubations and singleplex capture bead designs. Here, ruthenium hexamine liposomes had only mediocre performance so that low overall signal strength translated into higher LODs and worse specificity. A different marker such as ferroin may be an option in the future. The identification of further electrochemical markers will provide new opportunities for liposomes to function as multiplex, orthogonal or internal standard labels in electrochemical bioassays. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-024-05145-8.


Multiplex buffers
The buffer composition was investigated, as it has a large influence on DNA hybridization.With increasing stringency (e.g.lower salt concentration, higher formamide content) it becomes more difficult for mismatched DNA strands to hybridize until only perfectly matching sequences can hybridize.However, no effect on the non-specific signals was observed, when changing the composition of the hybridization buffer or washing buffer (Fig. SI7a).The stringency of the hybridization buffer was increased by decreasing the SSC concentration from 9× to 6× or 3×.However, only a general decrease in hybridization for all DNAs was observed, with no influence on the nonspecific signals.When increasing the stringency even further for the washing buffer by changing to 1xSSC and increasing the formamide content, again just an overall decrease of signals was observed, but no effect on the non-specific signals (Fig. SI7b).Other buffers like PBS or Tris-HCl also showed no improvements.These results were surprising at first, as decreasing signals from the liposomes with non-matching probes had been expected.This can only be explained by the fact, that all obtained signals result from correct hybridizations, which heavily supports the theory of reporter probe exchange between liposomes[29-32].In that case, matching reporter probes are carried by all liposomes including those intended as labels for other targets.

Hybridization specificity between probes and target
The interactions between capture probe and target (Fig. SI8a) and between reporter probe and target (Fig. SI8b) were investigated in more detail in separate experiments to confirm probe specificity.In both experiments the results can be summarized by two scenarios.Either the assay mixture contains a complete set consisting of matching probes, target and liposomes, which results in signals from all present liposomes, including unintended signals.Or there are no signals, if no matching set is present.
These results were also true, when using liposomes and probes against Cryptosporidium parvum from previous studies [20].Therefore, unwanted cross-hybridization caused by insufficient probe design could be excluded to be responsible for the non-specific signals.

One-step hybridization assays with two liposomes
Since RuHex liposomes showed consistently low signals and different behaviours in DLS measurements (data not shown), experiments with only two liposomes (one matching, one non-matching liposome) were carried out to investigate, whether just one liposome is causing problems resulting in the nonspecific signals.Therefore, all possible combinations just two liposomes were tested (Fig. SI9).In all cases, signals from both the matching and the non-matching liposome were obtained.Thus, it was concluded, that the non-specific signals are not caused by one liposome alone.

Assay time and liposome concentration
Most scenarios that could explain the non-specific binding require interaction between liposomes.Therefore, incubation time and liposome concentration were investigated (Fig. SI10).An incubation time of 10 min, 30 min or 90 min had no effect on the signal height.This also demonstrates a fast assay time.Decreasing the liposome concentration from 500 µmol L -1 to 50 µmol L -1 only led to the expected decrease in overall signals but had no effect on the non-specific signals.

Liposome fusion or aggregation
A potential aggregation or fusion of liposomes, that would explain non-specific signals, was investigated by DLS measurement (Fig. SI12).Measurements of individual liposomes were compared to mixtures of liposomes.No increase in liposome size between mixture and single measurements was observed, hinting at fusion or aggregation.In hybridization buffer all liposome peaks broadened, resulting in an increase in PdI (data not shown).It seems, that liposome morphology is affected in this buffer, but the non-specific signals also occurred in other buffers like HSS buffer, where this broadening effect was not observed.

Fig. S1
Fig. S1 LIG electrode used for all electrochemical measurements.It consits of LIG working and counter electrode and a pseudo Ag/AgCl reference electrode made with silver ink.The electrode area is restricted by nail polish.

Fig. S2
Fig. S2 Cross-reactivity study of methylene blue with FCN and mCL.Peak heights of 100 µmol L -1 methylene blue alone, in mixture with FCN and in mixture with mCL.The insert shows images the respective solution.The blue color decreases, when methylene blue gets reduced by FCN or mCL.

Fig. S4
Fig. S4 Hybridization assay test with new liposomes.Peak heights obtained from one-step, singleplex assays using only one liposome and its respective target DNA at concentrations of 0, 0.5, 25 nmol L -1 for a) InfB target and InfB/RuHex liposomes, b) Inf A target and Inf A/FCN liposomes and c) SC2 target and SC2/mCL liposomes.

Fig. S5
Fig. S5 Hybridization assays with liposomes with new encapsulant-reporter probe combinations.Liposomes were tested in one-step, multiplex assays with either one or all three targets present.And in singleplex assays with just their matching target present to confirm binding functionality.Tagret concentrations used were always 25 nmol L -1 .

Fig.
Fig. S8 a) Reporter probe specificity study.One-step multiplex assays with matching capture probe and target but different reporter probe modified liposomes b) Capture probe specificity study.One-step multiplex assays with different capture probe and target combinations or no capture probe.

Fig. S11
Fig. S11 Size by intensity determined through DLS measurements in HSS buffer of RuHex/Inf B, FCN/Inf A and mCL/SC2liposomes and a mixture of all three liposomes.

Fig. S12
Fig. S12 a) Multistep assays with separated liposome incubations in different orders.For every target the matching liposome was once incubated first and once incubated last.Target concentration was always 25 nmol L -1 .b) Influence of liposome incubation order in combination with MB mixed CPs or separated MB for every capture probe.Stepwise multiplex assays with all targets present at 25 nmol L -1 but different liposome incubation orders.Each liposome was used first once.Same orders were tested once with mixed MB/CPs and once with separated MB/CPs.

Fig. S13
Fig. S13 Studies on post-synthesis dual-modified liposomes.Target concentrations were always 25 nmol L-1.a) Peak heights from hybridization assays with RuHex/Inf B liposomes, RuHex/Inf B liposomes modified with more Inf B reporter probe using Inf B target and RuHex/Inf B liposomes modified with Inf A reporter probe tested against Inf A target.b) mCL/SC2 liposomes modified with varying amounts of additional Inf B reporter probe (0.1, 1, 10 pmol) and tested against Inf B target.c) FCN/Inf A liposomes with different ratios between total lipid concentration (10-500µmol L-1) and amount of Inf A reporter probe (6.8 -34 pmol) tested against Inf A target.

Fig. S14
Fig. S14 Comparison between multiplex assays with all three targets present and the sum of three individual, multiplex assays with just one target present.Target concentrations and liposome incubation order were kept the same.