How to Troubleshoot PEG Bioconjugation: Solubility, Conversion, Purification, and Characterization?

Explore PEG Reagents for Bioconjugation Needs

BOC Sciences offers functional PEG reagents for troubleshooting and optimizing PEG bioconjugation workflows, including Azide PEG, DBCO PEG, Maleimide PEG, NHS ester PEG, and Thiol PEG products for click chemistry, thiol coupling, amine modification, lipid-linked conjugation, and staged PEGylation designs.

Why PEG Bioconjugation Problems Happen?

PEG bioconjugation problems usually come from the interaction of several variables rather than a single incorrect reagent. PEG reagents introduce reactive chemistry, but they also change solubility, molecular size, hydrodynamic behavior, viscosity, surface hydration, and analytical response. A successful workflow must therefore control both chemical reactivity and physical behavior. The most useful troubleshooting approach is to separate the problem into reagent quality, substrate accessibility, reaction condition, purification, and characterization.

PEG Reagents Add Both Reactivity and Physicochemical Complexity

Functional PEG reagents are not passive spacers. They carry reactive end groups such as NHS ester, maleimide, thiol, azide, alkyne, DBCO, aldehyde, hydrazide, lipid anchor, or surface-reactive groups, and each end group has its own stability and reaction requirements. At the same time, the PEG chain changes the behavior of the reaction mixture. Longer PEGs can increase hydration and apparent molecular size, while lipid PEG, dye PEG, DBCO PEG, and multi-arm PEG may introduce hydrophobic regions, aggregation tendency, adsorption, or broader chromatographic behavior. PEG molecular weight distribution can further complicate mass analysis and peak shape. Troubleshooting should therefore evaluate both the terminal chemistry and the PEG chain structure.

A Chemically Matched Reaction Can Still Fail Experimentally

Functional group pairing is only the first requirement. NHS ester PEG can react with primary amines, but the ester may hydrolyze before productive coupling if the pH, buffer, or timing is unsuitable. Maleimide PEG can react with thiols, but thiol oxidation, high pH, reducing agents, or hidden cysteines may reduce conversion. Azide PEG and Alkyne PEG can support CuAAC, but copper catalyst compatibility, ligand choice, and residual copper removal may limit practical use. DBCO PEG can support copper-free SPAAC, but DBCO bulk and hydrophobicity can create solubility or accessibility issues. A reaction can be chemically correct but experimentally inefficient if the full system is not compatible.

Troubleshooting Should Separate Reaction Failure from Purification or Analysis Failure

It is important to distinguish whether the conjugate failed to form, formed but degraded, formed but was lost during purification, or formed but was not detected by the chosen analytical method. Low apparent yield may reflect poor conversion, but it may also reflect poor recovery, adsorption to filtration membranes, broad co-elution with free PEG, or an analytical method that cannot resolve PEGylated species. For example, a PEGylated protein may migrate abnormally on SDS-PAGE, and a PEG-lipid conjugate may produce strong signal from residual free PEG-lipid rather than covalently associated product. Troubleshooting should identify the specific failure point before changing reagent ratio or reaction time.

Solubility and Aggregation Problems in PEG Bioconjugation

PEG is often chosen to improve aqueous compatibility, but not every PEG derivative behaves like a simple water-soluble polymer. Reactive end groups, hydrophobic anchors, dyes, strained alkynes, long chains, high concentration, salts, and substrate properties can all cause turbidity, precipitation, phase separation, or aggregation. Solubility troubleshooting should be performed under the actual reaction conditions rather than only in the stock solvent.

PEG Reagent Dissolves in Stock Solvent but Precipitates in Reaction Buffer

A PEG reagent may dissolve well in DMSO, DMF, water, or a concentrated stock solution but become cloudy after dilution into the final buffer. This can happen when salt concentration is high, pH changes the charge state of the substrate, the final concentration is too high, or the PEG reagent contains hydrophobic groups such as DBCO, lipid anchors, aromatic dyes, PLGA, PLA, or long hydrocarbon chains. Rapid addition of a concentrated stock can create local high concentrations that trigger temporary or permanent aggregation. The first check should be a small-scale compatibility test using the actual buffer, pH, salt concentration, co-solvent percentage, and reaction concentration. If turbidity appears before adding the target molecule, the PEG reagent and reaction medium are already mismatched.

Long PEG, Lipid PEG, Dye PEG, and DBCO PEG Can Behave Differently

Long PEG chains and specialized PEG derivatives can behave differently from short monodisperse spacers. Lipid PEG derivatives can form micelles or interfacial aggregates. DBCO-bearing PEGs may show stronger hydrophobic interaction than azide- or alkyne-bearing PEGs. Dye PEGs can aggregate through aromatic stacking or hydrophobic clustering. Multi-Arm PEG can increase viscosity, local functionality, and network formation risk. In troubleshooting, the PEG derivative should be evaluated as a whole structure rather than only by its reactive end group. A longer or more hydrophilic PEG may improve some systems, but a shorter monodisperse PEG may be better when broad distribution, viscosity, or purification behavior is the main problem.

Substrate Precipitation After PEG Addition

Substrate precipitation after PEG addition can occur even when the PEG reagent itself remains soluble. Proteins may precipitate if the buffer pH approaches the isoelectric region, if organic solvent is added too quickly, if local PEG concentration is high, or if overmodification changes surface charge and hydration. Peptides and small molecules may precipitate when PEGylation changes hydrophobic balance or when salt and co-solvent conditions are not compatible. Nanoparticles may aggregate if PEG density, ionic strength, or surface charge changes abruptly. Oligonucleotides may show altered solubility or retention if hydrophobic DBCO, lipid, or dye modules are introduced. The troubleshooting priority is to identify whether precipitation occurs immediately upon PEG addition, during reaction, during quenching, or during purification, because each stage suggests a different cause.

Practical Strategies to Improve Solubility Without Damaging the Substrate

Solubility can often be improved by lowering reaction concentration, pre-diluting the PEG stock, adding the PEG reagent slowly with mixing, reducing salt concentration, changing buffer identity, adjusting pH away from precipitation-prone regions, or screening compatible co-solvent percentages. For sensitive proteins and oligonucleotides, the co-solvent level should remain low enough to preserve structure and function. For hydrophobic PEG derivatives, a more hydrophilic spacer, shorter hydrophobic module, or different reaction order may be needed. If DBCO or lipid groups are driving aggregation, switching to an azide-bearing intermediate followed by a better-controlled final ligation may help. When prior data are unavailable, small-scale solubility screening should be completed before committing valuable substrate.

Low Conversion and Poor Coupling Efficiency

Low conversion can be caused by inactive PEG reagent, inaccessible substrate functional groups, incorrect pH, incompatible buffer, insufficient mixing, poor solubility, or excessive steric hindrance. The most common mistake is to increase PEG equivalents before confirming that the reagent end group is active and that the target functional group is accessible. A controlled troubleshooting sequence should first verify reagent integrity and substrate availability, then optimize reaction variables one at a time.

Inactive or Degraded PEG End Groups

Reactive PEG end groups can lose activity during storage, handling, or exposure to moisture. NHS esters can hydrolyze, maleimides can hydrolyze or become less selective, thiols can oxidize to disulfides, aldehydes can hydrate or oxidize, and some click handles can suffer from contamination, adsorption, or poor handling. Thiol PEG reagents are particularly sensitive to oxidation, while activated esters are sensitive to water and prolonged exposure to aqueous buffer. Before changing reaction conditions, confirm reagent identity, storage temperature, container history, opening frequency, purity, and end-group conversion when possible. Fresh reagent or a small positive-control reaction can often distinguish end-group inactivity from substrate-related problems.

Functional Groups Are Present but Not Accessible

A substrate may contain the correct functional group but still react poorly because the group is sterically hidden or chemically unavailable. Lysine residues on proteins may be buried in folded regions, cysteines may form disulfides, azide or alkyne handles may be close to a surface, and particle-bound groups may be masked by a dense coating. For antibodies, peptides, enzymes, oligonucleotides, or surfaces, the apparent number of functional groups does not always equal the number of reactive groups. Accessibility can sometimes be improved by using a longer PEG spacer, lowering surface density, changing reaction order, introducing a more exposed handle, or selecting Heterobifunctional PEG linkers for staged modification. If accessibility is the bottleneck, adding more reagent may increase background without improving useful conjugation.

pH, Buffer, and Co-Solvent Mismatch

Each conjugation chemistry has a suitable pH and buffer window. NHS ester reactions require a balance between amine nucleophilicity and ester hydrolysis. Maleimide-thiol coupling usually requires conditions that keep thiols reactive while limiting maleimide hydrolysis and nonspecific reaction. CuAAC requires copper, ligand, and often a reducing system that may not be compatible with every biomolecule. SPAAC avoids copper but may be slower if azide or DBCO groups are poorly accessible. Carbonyl-directed coupling may require mildly acidic conditions and may proceed slowly without stabilization. Buffers containing primary amines can compete with NHS ester PEG, thiol-containing additives can compete with maleimide PEG, and chelators can interfere with CuAAC. Buffer choice should be treated as a reaction variable, not a passive background component.

Insufficient Reagent Ratio or Poor Mixing

PEG reagent ratio and mixing quality can strongly affect conversion. High-molecular-weight PEGs diffuse more slowly than small molecules, and viscous or crowded mixtures may reduce effective collision frequency. Insufficient PEG equivalents can lead to incomplete modification, while poor mixing can create local overconcentration, precipitation, or uneven reaction. The optimal ratio depends on substrate concentration, number of available sites, desired modification degree, PEG molecular weight, and purification capacity. When optimizing, change one factor at a time: PEG equivalents, concentration, reaction time, temperature, pH, co-solvent level, or addition order. This makes it easier to interpret whether conversion improves because of chemical conditions or simply because the system becomes more soluble.

When Increasing PEG Excess Helps — and When It Makes Things Worse

Increasing PEG excess can improve conversion when the reactive group is active, the substrate is accessible, and purification can remove free PEG. However, excessive PEG can make the workflow worse by increasing free PEG contamination, overmodification, aggregation, viscosity, cost, and purification burden. In protein and antibody workflows, high PEG excess may produce broad modification distributions. In oligonucleotide and peptide workflows, excess PEG may overlap with product peaks. In particle and surface workflows, excess PEG may adsorb noncovalently and create false-positive loading signals. The best strategy is to define the desired modification degree and purification method before increasing equivalents. If low conversion persists despite reasonable excess, the root cause is likely accessibility, solubility, end-group inactivity, or condition mismatch.

Reaction-Specific Troubleshooting: NHS, Maleimide, Click, and Carbonyl Chemistry

Different PEG conjugation reactions fail in different ways. Troubleshooting should therefore be specific to the reaction class rather than applying one general fix to every system. NHS ester chemistry, maleimide-thiol coupling, click chemistry, carbonyl-directed ligation, and surface PEGylation each have characteristic failure modes and optimization points.

NHS Ester PEG Troubleshooting

NHS ester PEG problems often involve hydrolysis, unsuitable pH, competing amines, or uncontrolled multi-site modification. If pH is too low, primary amines may be insufficiently nucleophilic; if pH is too high or reaction time is too long, NHS ester hydrolysis increases. Buffers containing primary amines, such as Tris or glycine-containing systems, can compete with the target substrate. For proteins, multiple lysines may generate heterogeneous products, while for small molecules and peptides, incomplete solubility can limit conversion. Practical adjustments include using a non-amine buffer, preparing fresh PEG stock, controlling pH and reaction time, avoiding prolonged aqueous preincubation, lowering or increasing PEG equivalents according to desired modification degree, and planning purification to remove hydrolyzed PEG and unreacted reagent.

Maleimide PEG and Thiol PEG Troubleshooting

Maleimide-thiol systems are sensitive to thiol availability, pH, oxidation, and competing nucleophiles. Low conversion may occur if thiols are oxidized, if the target cysteine is buried, if maleimide has hydrolyzed, or if reducing agents and buffer additives interfere. Thiol PEG itself can oxidize to disulfide-containing impurities, reducing effective concentration. Reaction pH should support thiol reactivity while avoiding conditions that reduce maleimide selectivity or stability. Before troubleshooting with more reagent, verify that the thiol substrate is reduced and accessible, that maleimide PEG is fresh, and that the buffer does not contain unintended thiol competitors. For sensitive systems, staged reaction and rapid purification can reduce side reactions.

Azide, Alkyne, and DBCO PEG Troubleshooting

Click PEG troubleshooting depends on whether the reaction is CuAAC or SPAAC. CuAAC between azide and alkyne groups can be efficient but requires compatible copper source, ligand, reducing system, oxygen control, and metal-removal strategy. Poor conversion may result from inaccessible handles, incompatible buffers, chelators, insufficient ligand, or copper-sensitive substrates. SPAAC between azide and DBCO or BCN avoids copper, but it may be slower in crowded systems and can suffer from DBCO hydrophobicity, steric hindrance, or nonspecific adsorption. If CuAAC gives low conversion, check catalyst composition before increasing PEG. If SPAAC gives low conversion, evaluate handle accessibility, DBCO solubility, PEG length, and whether a more exposed azide or strained alkyne position is needed.

Aldehyde, Hydrazide, and Carbonyl-Directed PEG Troubleshooting

Carbonyl-directed PEG coupling can be useful for aldehyde-bearing substrates, oxidized glycans, oxidized polysaccharides, and hydrazide or aminooxy ligation routes, but reaction conditions require careful control. Aldehyde PEG and Hydrazide PEG reactions may be slow, pH-sensitive, or reversible depending on linkage type. Glycan oxidation can be insufficient, producing low conversion, or excessive, damaging the substrate. Some carbonyl-based linkages may require reduction or stabilization if long-term linkage stability is needed in the research workflow. Troubleshooting should confirm carbonyl generation, avoid over-oxidation, adjust pH, manage reaction time, and evaluate whether the product can be purified without reversing or degrading the linkage.

Surface and Nanoparticle PEGylation Troubleshooting

Surface and nanoparticle PEGylation can be difficult because apparent attachment may come from adsorption rather than covalent reaction. Surface functional group density may be lower than expected, PEG may adsorb noncovalently, and washing may remove weakly associated material. Particles may aggregate after PEG addition if surface charge, salt concentration, PEG density, or ligand hydrophobicity changes. Zeta potential may not shift clearly if the PEG layer is neutral or if the surface is already shielded. For surfaces and particles, troubleshooting should include control samples, aggressive but appropriate washing, ligand quantification, DLS, zeta potential, contact angle, fluorescence only with adsorption controls, and comparison before and after purification. Surface Modification and Functionalization workflows should distinguish covalent PEGylation from trapped or adsorbed PEG reagent.

Purification Problems: Free PEG, Partial Products, and Low Recovery

Many PEG bioconjugation workflows fail during purification rather than during reaction. Free PEG can be difficult to remove, partially modified products can overlap with desired product, and the final conjugate can be lost through adsorption, precipitation, membrane retention, or broad elution. Purification should therefore be planned before setting up the reaction, especially when using high-molecular-weight PEG, polydisperse PEG, PEG-lipids, dye PEG, or multifunctional PEG reagents.

Why Free PEG Is Difficult to Remove

Free PEG can be difficult to remove because it is highly hydrated, flexible, and sometimes similar in size or polarity to the target product. High-molecular-weight free PEG may co-elute with proteins during SEC, while small PEG reagents may overlap with salts, small molecules, or peptide products in HPLC. Polydisperse PEG creates a distribution of species rather than one clean impurity peak. PEG-lipid and dye PEG impurities may associate with particles, membranes, or column materials. If free PEG remains after purification, it can interfere with quantification, functional assays, fluorescence readouts, viscosity, and downstream conjugation. The purification method should be selected based on the most difficult impurity, not only on the desired product.

Separating Unmodified, Partially Modified, and Overmodified Products

PEGylation often produces a distribution of products, especially when multiple reactive sites are present. Protein and antibody samples may contain unmodified, singly modified, and multiply modified species. Oligonucleotide samples may contain unmodified strand, partially reacted product, and excess linker. Surface and particle samples may contain covalently attached PEG, adsorbed PEG, and free PEG in solution. Separation can be based on size, charge, hydrophobicity, affinity, or mobility. SEC may separate by hydrodynamic size, IEX by charge, HIC by hydrophobicity, HPLC by retention, PAGE by mobility, and CE by electrophoretic behavior. Method choice should reflect how the PEG modification changes the product relative to impurities.

Choosing Purification Methods by Conjugate Type

Small molecules and short peptide PEG conjugates are often purified by HPLC and confirmed by LC-MS or NMR. Protein and antibody conjugates may require SEC, IEX, HIC, desalting, ultrafiltration, or electrophoretic analysis. Oligonucleotide conjugates may require ion-pair HPLC, anion exchange HPLC, PAGE, desalting, or ultrafiltration. Lipid PEG and nanoparticle systems may require dialysis, filtration, centrifugation, SEC, washing, or formulation-specific cleanup. Surface-bound systems require washing and surface analysis rather than conventional solution purification. When purification is difficult, changing PEG length, switching to Monodisperse PEG, reducing excess reagent, or using a staged heterobifunctional PEG linkers for stepwise bioconjugation strategy may improve resolution.

Low Recovery After Purification

Low recovery can occur even when conversion and separation are acceptable. PEG conjugates may adsorb to membranes, tubing, vials, columns, or particle surfaces. They may precipitate during buffer exchange, become unstable after dilution, bind strongly to chromatographic media, or be retained by ultrafiltration membranes. Protein and peptide conjugates may lose function if exposed to harsh elution conditions or long processing time. Lipid and nanoparticle conjugates may be lost during centrifugation, filtration, or washing. Troubleshooting recovery should compare mass balance before and after each purification step. Adjusting buffer composition, reducing processing time, adding compatible stabilizing components, changing membrane material, modifying pH or salt, and avoiding unnecessary concentration steps can improve recovery.

Purification Should Be Planned Before Reaction Setup

Purification planning should influence reagent selection from the beginning. A high-molecular-weight PEG may improve solubility but make free PEG removal difficult. A DBCO-bearing linker may enable copper-free ligation but increase hydrophobic adsorption. A polydisperse PEG may be acceptable for surface shielding but unsuitable for exact mass confirmation. A large excess of PEG may improve conversion but overwhelm purification. Before the reaction begins, decide which impurities will be present and how they will be removed. If the intended product cannot be separated from free PEG, unmodified substrate, or partial products with available methods, the reagent, PEG length, or reaction route should be redesigned.

Characterization Problems and Misleading Analytical Results

PEG conjugate characterization can be misleading because PEG changes apparent size, hydration, chromatographic behavior, electrophoretic mobility, and mass spectral response. A single analytical method rarely provides enough evidence. The goal of characterization is not only to show that a conjugate exists, but also to confirm identity, purity, modification degree, free PEG removal, aggregation state, and functional performance.

PEG Changes Apparent Molecular Size and Migration Behavior

PEG has a large hydrated volume relative to its dry molecular weight, so PEGylated products may appear larger than expected by SEC, DLS, SDS-PAGE, or CE. A PEGylated protein may migrate more slowly on SDS-PAGE than its true mass increase would suggest. A PEGylated particle may show increased hydrodynamic diameter without a proportional increase in dry mass. A surface bearing PEG may show changes in contact angle or protein adsorption without a strong elemental signal. These behaviors are not necessarily errors, but they must be interpreted correctly. Troubleshooting should compare PEG-modified samples with unmodified controls, free PEG controls, and orthogonal measurements.

Polydisperse PEG Broadens Signals and Complicates Mass Analysis

Polydisperse PEG produces a distribution of chain lengths, which can broaden HPLC peaks, complicate mass spectra, and make exact structural assignment difficult. This is especially problematic for small-molecule, peptide, and oligonucleotide conjugates where precise mass confirmation is expected. In contrast, high-molecular-weight polydisperse PEG may be acceptable in applications focused on surface hydration or steric shielding, provided that the product can be characterized by suitable methods. If mass analysis is central to the project, monodisperse PEG should be considered. If polydisperse PEG is already used, characterization may require SEC/GPC, NMR end-group analysis, MALDI-type methods, or indirect quantification rather than relying on a single exact mass peak.

One Analytical Method Is Rarely Enough

Orthogonal characterization is often required because each method answers a different question. HPLC may show purity but not functional activity. LC-MS may confirm small conjugates but may struggle with high-molecular-weight PEG distributions. SEC can show size distribution but not exact modification site. IEX can resolve charge variants but may not distinguish free PEG. SDS-PAGE can compare mobility but may misrepresent size. DLS can reveal aggregation but cannot prove covalent attachment. Zeta potential, contact angle, fluorescence, ligand quantification, and surface methods can support interface analysis but need controls. Robust troubleshooting combines methods that address identity, purity, loading, aggregation, residual reagent, and function.

Distinguishing True Conjugation from Adsorption or Residual Reagent

Adsorption and residual reagent can create false-positive results, especially in fluorescent PEG, biotin PEG, PEG-lipid, nanoparticle, and surface workflows. A strong fluorescence signal may come from trapped dye PEG rather than covalent conjugation. A biotin assay may detect free biotin PEG that was not fully removed. A DLS size increase may reflect adsorbed PEG-lipid micelles rather than stable surface modification. Surface fluorescence can remain after weak adsorption if washing is insufficient. Troubleshooting should include no-reactive-group controls, no-click controls, free PEG controls, stronger washing tests, and analytical methods that distinguish covalent product from noncovalent carryover.

Characterization Should Confirm Identity, Purity, Loading, and Function

A complete characterization strategy should answer four questions: what is the product, how pure is it, how much PEG or ligand is attached, and does it still perform the intended function. For soluble conjugates, this may involve HPLC, LC-MS, NMR, SEC, IEX, HIC, SDS-PAGE, CE, UV-vis, fluorescence, or activity tests. For particles and surfaces, this may involve DLS, zeta potential, contact angle, fluorescence imaging, ligand quantification, and adsorption controls. PEGylation Analysis and Method Verification can be useful when analytical ambiguity is the main obstacle rather than reaction conversion itself.

Structured Troubleshooting Workflow for PEG Bioconjugation

A structured troubleshooting workflow helps prevent random condition changes that make results harder to interpret. The following sequence moves from reagent verification to reaction conditions, purification, characterization, and redesign. It can be applied to amine-reactive, thiol-reactive, click, carbonyl-directed, lipid-linked, and surface PEGylation workflows. For upstream reagent choice, see this PEG reagent selection guide.

Fig. 2. Workflow for troubleshooting PEG bioconjugation problems (BOC Sciences Authorized).

How BOC Sciences Supports PEG Bioconjugation Troubleshooting?

BOC Sciences supports troubleshooting for PEG bioconjugation and PEGylation workflows involving proteins, antibodies, peptides, oligonucleotides, small molecules, lipids, nanoparticles, and functional surfaces. Support can focus on reagent selection, end-group activity, solubility screening, reaction condition optimization, purification strategy, and analytical method verification. The goal is to identify the real source of the issue and improve the workflow without unnecessary trial-and-error.

PEG Reagent Quality and End-Group Evaluation

• Evaluate PEG reagent type, end group, molecular weight, dispersity, storage history, and handling conditions.
• Assess risks from NHS ester hydrolysis, maleimide instability, thiol oxidation, aldehyde reactivity loss, and click handle incompatibility.
• Support comparison of azide, alkyne, DBCO, maleimide, thiol, NHS ester, aldehyde, hydrazide, lipid, and multi-arm PEG options.
• Help determine whether poor results are caused by reagent quality, substrate accessibility, or reaction condition mismatch.

Solubility and Reaction Condition Optimization

• Optimize buffer, pH, salt concentration, co-solvent ratio, PEG equivalents, reaction concentration, addition order, time, and temperature.
• Troubleshoot turbidity, precipitation, aggregation, low conversion, substrate instability, and reaction-to-reaction variability.
• Support PEG length screening to balance solubility, steric accessibility, purification behavior, and analytical clarity.
• Recommend route adjustments when DBCO, lipid PEG, dye PEG, long PEG, or multifunctional PEG causes handling difficulties.

Purification Strategy Development

• Recommend purification strategies for small molecules, peptides, proteins, antibodies, oligonucleotides, lipids, particles, and surfaces.
• Support HPLC, SEC, IEX, HIC, PAGE, CE, desalting, ultrafiltration, dialysis, filtration, centrifugation, and washing workflows.
• Help remove free PEG, unmodified substrate, partial products, overmodified products, salts, residual catalyst, and adsorbed reagent.
• Improve purification planning by matching PEG size, product type, impurity profile, and available analytical methods.

Characterization and Method Verification

• Support HPLC, LC-MS, NMR, SEC/GPC, SDS-PAGE, CE, DLS, zeta potential, contact angle, ligand quantification, and fluorescence analysis.
• Help confirm identity, purity, modification degree, residual free PEG, aggregation state, surface attachment, and functional performance.
• Design orthogonal characterization strategies when one method gives broad, ambiguous, or misleading results.
• Support method verification for PEG conjugates where product behavior differs from unmodified substrates.